Thermolysin variants

ABSTRACT

The present invention provides methods and compositions comprising at least one thermolysin-like neutral protease enzyme with improved storage stability and/or catalytic activity. In some embodiments, the thermolysin finds use in cleaning and other applications comprising detergent. In some particularly preferred embodiments, the present invention provides methods and compositions comprising thermolysin formulated and/or engineered to resist detergent-induced inactivation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.14/794,687, filed Jul. 8, 2015, which is a Divisional of U.S.application Ser. No. 14/035,441, filed on Sep. 24, 2013, which is aDivisional of U.S. application Ser. No. 12/740,782, filed Dec. 22, 2010,now U.S. Pat. No. 8,569,034, which is a 371 National Phase applicationof PCT/US2008/012276, filed Oct. 28, 2008, which claims the benefit ofU.S. Provisional Application No. 60/984,664, filed on Nov. 1, 2007, thedisclosure of each application is incorporated herein in its entirety.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file20180426_NB31056USDIV3_SeqLst.txt, created on Apr. 26, 2018, which is 20kb in size.

FIELD OF THE INVENTION

The present invention provides methods and compositions comprising atleast one thermolysin-like neutral protease enzyme with improved storagestability and/or catalytic activity. In some embodiments, thethermolysin finds use in cleaning and other applications comprisingdetergent. In some particularly preferred embodiments, the presentinvention provides methods and compositions comprising thermolysinformulated and/or engineered to resist detergent-induced inactivation.

BACKGROUND OF THE INVENTION

Bacilli are gram-positive bacteria that secrete a number of industriallyuseful enzymes, which can be produced cheaply in high volume byfermentation. Examples of secreted Bacillus enzymes are the subtilisinserine proteases, zinc containing neutral proteases, alpha-amylases, andcellulases. Bacillus proteases are widely used in the textile, laundryand household industries (Galante, Current Organic Chemistry,7:1399-1422, 2003; and Showell, Handbook of Detergents, Part D:Formulation, Hubbard (ed.), NY: Taylor and Francis Group, 2006). Highlyefficient color and stain removal from laundry require proteases.However, liquid preparations of cleaning and washing reagents typicallycontain builders, surfactants, and metal chelators, which have adestabilizing effect on most proteases.

Detergent and other cleaning compositions typically include a complexcombination of active ingredients. For example, most cleaning productsinclude a surfactant system, enzymes for cleaning, bleaching agents,builders, suds suppressors, soil-suspending agents, soil-release agents,optical brighteners, softening agents, dispersants, dye transferinhibition compounds, abrasives, bactericides, and perfumes. Despite thecomplexity of current detergents, there are many stains that aredifficult to completely remove. Furthermore, there is often residuebuild-up, which results in discoloration (e.g., yellowing) anddiminished aesthetics due to incomplete cleaning. These problems arecompounded by the increased use of low (e.g., cold water) washtemperatures and shorter washing cycles. Moreover, many stains arecomposed of complex mixtures of fibrous material, mainly incorporatingcarbohydrates and carbohydrate derivatives, fiber, and cell wallcomponents (e.g., plant material, wood, mud/clay based soil, and fruit).These stains present difficult challenges to the formulation and use ofcleaning compositions.

In addition, colored garments tend to wear and show appearance losses. Aportion of this color loss is due to abrasion in the laundering process,particularly in automated washing and drying machines. Moreover, tensilestrength loss of fabric appears to be an unavoidable result ofmechanical and chemical action due to use, wearing, and/or washing anddrying. Thus, a means to efficiently and effectively wash coloredgarments so that these appearance losses are minimized is needed.

In sum, despite improvements in the capabilities of cleaningcompositions, there remains a need in the art for detergents that removestains, maintain fabric color and appearance, and prevent dye transfer.In addition, there remains a need for detergent and/or fabric carecompositions that provide and/or restore tensile strength, as well asprovide anti-wrinkle, anti-bobbling, and/or anti-shrinkage properties tofabrics, as well as provide static control, fabric softness, maintainthe desired color appearance, and fabric anti-wear properties andbenefits. In particular, there remains a need for the inclusion ofcompositions that are capable of removing the colored components ofstains, which often remain attached to the fabric being laundered. Inaddition, there remains a need for improved methods and compositionssuitable for textile bleaching.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprising atleast one thermolysin-like neutral protease enzyme with improved storagestability and/or catalytic activity. In some embodiments, thethermolysin finds use in cleaning and other applications comprisingdetergent. In some particularly preferred embodiments, the presentinvention provides methods and compositions comprising thermolysinformulated and/or engineered to resist detergent-induced inactivation.

The present invention provides compositions comprising an isolatedthermolysin and a neutral metalloprotease inhibitor, wherein thethermolysin is a Geobacillus thermolysin or a Bacillus thermolysin. Insome embodiments, the compositions of the invention comprise an isolatedthermolysin and a neutral metalloprotease inhibitor chosen fromphosphoramidon and galardin. In some embodiments, the thermolysin of thecompositions of the invention is a G. caldoproteolyticus or a G.stearothermophilus, thermolysin, while in other embodiments, thethermolysin of the compositions of the invention is a B.thermoproteolyticus thermolysin. In some particular embodiments, thecompositions of the invention comprise a thermolysin has at least 50%(50 to 100%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%) amino acid identity with the thermolysincomprising the amino acid sequence set forth in SEQ ID NO:3. In someother embodiments, the compositions of the invention comprise athermolysin that comprises the amino acid sequence set forth in SEQ IDNO:3. In yet other embodiments, the compositions of the inventioncomprise a thermolysin of SEQ ID NO:3.

In addition the present invention provides an isolated thermolysinvariant having improved stability and/or performance. In some preferredembodiments, the thermolysin variant is a Geobacillus thermolysinvariant having an amino acid sequence comprising one or moresubstitutions at positions chosen from positions equivalent to positions6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280of the amino acid sequence set forth as SEQ ID NO:3. In a subset ofthese embodiments, the one or more substitutions comprise one, two,three, four or five substitutions at positions chosen from positionsequivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156,196, 273, 278, and 280 of the amino acid sequence set forth as SEQ IDNO:3. In further embodiments, the invention provides an isolatedGeobacillus thermolysin variant having an amino acid sequence comprisingone or more substitutions at positions chosen from positions equivalentto positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128,129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of theamino acid sequence set forth as SEQ ID NO:3, and having improvedstability and/or performance. In a subset of these embodiments, the oneor more substitutions comprise one, two, three, four or fivesubstitutions at positions chosen from positions equivalent to positions4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128, 129, 151,156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of the amino acidsequence set forth as SEQ ID NO:3.

In some other embodiments, the invention provides a thermolysin variantthat comprises one or more substitutions chosen from the group of thesubstitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P, T006Q,T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P,V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N,T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K,S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y,Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V,Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K,T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M,T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V,Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H,Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S,T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q,T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E,S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y,Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as listed in Table 7-1,Table 8-1 and Table 8-2, and that has improved stability and/orperformance. In a subset of these embodiments, the one or moresubstitutions comprise one, two, three, four or five substitutionschosen from the group of substitutions T006G, T006H, T006I, T006K,T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y, V007F, V007H,V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y, T049G, T049H,T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I, A058P, A058R,F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I,Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N,Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T, I156W, G196R,Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R, T006A, T006C,T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R, A056Y, A058S,S065C, S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T,Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q,Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K, I156M, I156R,I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C,T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L, N280M, N280S,T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C, A058E, Q061E,Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I,Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, andI156E, as listed in Table 7-1, Table 8-1 and Table 8-2.

Moreover the present invention provides an isolated thermolysin varianthaving improved stability and/or performance as compared to wild-typeGeobacillus sp. thermolysin (e.g., thermolysin comprising the amino acidsequence set forth as SEQ ID NO:3). In some embodiments, the inventionprovides an isolated thermolysin variant having improvements thatcomprise one or more of improved thermostability, improved performanceunder lower or higher pH conditions, and improved autolytic stability.

In some embodiments, the invention provides a Bacillus sp. host celltransformed with a polynucleotide encoding a thermolysin variant having50 to 99% (at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%) amino acid identity with the amino acid sequence ofSEQ ID NO:3.

Also provided by the present invention are methods for producing anenzyme having thermolysin activity, comprising: i) transforming a hostcell with an expression vector comprising a polynucleotide encoding athermolysin variant having 50 to 99% identity (at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid identitywith the thermolysin comprising the amino acid sequence set forth in SEQID NO:3, and ii) cultivating the transformed host cell under conditionssuitable for the production of the thermolysin. Optionally, the methodof the invention further comprises harvesting the produced thermolysin.In some embodiments, the invention provides for methods for producing anenzyme having thermolysin activity, comprising: i) transforming a hostcell with an expression vector comprising a polynucleotide encoding athermolysin variant having polynucleotide encoding the thermolysinvariant has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% amino acid identity with the thermolysin comprising theamino acid sequence set forth in SEQ ID NO:3, and ii) cultivating thetransformed host cell under conditions suitable for the production ofthe thermolysin. Optionally, the methods further comprise the step ofharvesting the produced thermolysin. In some other embodiments, theinvention provides a method for producing an enzyme having thermolysinactivity, comprising: i) transforming a Bacillus species (e.g., Bsubtilis) host cell with an expression vector comprising apolynucleotide encoding a thermolysin variant having 50 to 99% identity(at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%) amino acid identity with the thermolysin comprising the amino acidsequence set forth in SEQ ID NO:3, and ii) cultivating the transformedhost cell under conditions suitable for the production of thethermolysin. Optionally, the methods further comprise the step ofharvesting the produced thermolysin.

In some embodiments, the present invention provides compositionscomprising at least one thermolysin variant obtained from therecombinant Bacillus sp. host cell of the present invention. In someembodiments, the composition comprising at least one thermolysin variantfurther comprises at least one calcium ion and/or zinc ion. In somealternative embodiments, the composition comprising at least onethermolysin variant further comprises at least one stabilizer. In asubset of these embodiments, the stabilizer is chosen from borax,glycerol, zinc ions, calcium ions, and calcium formate. In someembodiments, the stabilizer is at least one competitive inhibitor thatstabilizes the thermolysin in the presence of an anionic surfactant.Alternatively, the compositions comprising at least one thermolysinvariant, comprise at least one calcium ion and/or zinc ion, incombination with at least one stabilizer. Any one of the stabilizersrecited above may be combined with the at least one calcium ion and/orzinc ion to provide the compositions comprising at least one thermolysinvariant. In a subset of these embodiments, the stabilizer is chosen fromborax, glycerol, zinc ions, calcium ions, and calcium formate. In someother embodiments, the stabilizer is at least one competitive inhibitorthat stabilizes the thermolysin in the presence of an anionicsurfactant.

In other embodiments, the invention provides a composition comprising atleast one thermolysin variant obtained from the recombinant Bacillus sp.host cell of the present invention, in combination with at least oneadditional enzyme or enzyme derivative chosen from proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases. In some embodiments, the compsitoncomprising at least one thermolysin variant and at least one additionalenzyme or enzyme derivative chosen from proteases, amylases, lipases,mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, andcellulases further comprises at least one stabilizer. In a subset ofthese embodiments, the stabilizer is chosen from borax, glycerol, zincions, calcium ions, and calcium formate. In some embodiments, thestabilizer is at least one competitive inhibitor that stabilizes thethermolysin in the presence of an anionic surfactant. Alternatively, thecompositions comprising at least one thermolysin variant, comprise atleast one calcium ion and/or zinc ion, in combination with at least onestabilizer. Any one of the stabilizers recited above may be combinedwith the at least one calcium ion and/or zinc ion to provide thecompositions comprising at least one thermolysin variant. In a subset ofthese embodiments, the stabilizer is chosen from borax, glycerol, zincions, calcium ions, and calcium formate. In some other embodiments, thestabilizer is at least one competitive inhibitor that stabilizes thethermolysin in the presence of an anionic surfactant.

In some embodiments, present invention provides a cleaning compositioncomprising at least one thermolysin variant obtained from therecombinant Bacillus sp. host cell of the present invention. In someembodiments, the cleaning composition comprising at least onethermolysin variant, further comprises at least one calcium ion and/orzinc ion. In some alternative embodiments, the cleaning compositioncomprising at least one thermolysin variant, further comprises at leastone stabilizer. In a subset of these embodiments, the stabilizer ischosen from borax, glycerol, zinc ions, calcium ions, and calciumformate. In some embodiments, the stabilizer is at least one competitiveinhibitor that stabilizes the thermolysin in the presence of an anionicsurfactant. Alternatively, the cleaning compositions comprising at leastone thermolysin variant, comprise at least one calcium ion and/or zincion, in combination with at least one stabilizer. Any one of thestabilizers recited above may be combined with the at least one calciumion and/or zinc ion to provide the compositions comprising at least onethermolysin variant. In some embodiments, the stabilizer is at least onecompetitive inhibitor that stabilizes the thermolysin in the presence ofan anionic surfactant.

In other embodiments, the invention provides a cleaning compositioncomprising at least one thermolysin variant obtained from therecombinant Bacillus sp. host cell of the present invention, incombination with at least one additional enzyme or enzyme derivativechosen from proteases, amylases, lipases, mannanases, pectinases,cutinases, oxidoreductases, hemicellulases, and cellulases. In someembodiments, the cleaning compsiton comprising at least one thermolysinvariant and at least one additional enzyme or enzyme derivative chosenfrom proteases, amylases, lipases, mannanases, pectinases, cutinases,oxidoreductases, hemicellulases, and cellulases further comprises atleast one stabilizer. In a subset of these embodiments, the stabilizeris chosen from borax, glycerol, zinc ions, calcium ions, and calciumformate. In some embodiments, the stabilizer is at least one competitiveinhibitor that stabilizes the thermolysin in the presence of an anionicsurfactant. Alternatively, the cleaning compositions comprising at leastone thermolysin variant, comprise at least one calcium ion and/or zincion, in combination with at least one stabilizer. Any one of thestabilizers recited above may be combined with the at least one calciumion and/or zinc ion to provide the compositions comprising at least onethermolysin variant. In a subset of these embodiments, the stabilizer ischosen from borax, glycerol, zinc ions, calcium ions, and calciumformate. In some other embodiments, the stabilizer is at least onecompetitive inhibitor that stabilizes the thermolysin in the presence ofan anionic surfactant.

In some embodiments, the present invention provides compositionscomprising an isolated thermolysin variant having improved stabilityand/or performance. In some embodiments, the composition comprising theisolated thermolysin variant having improved stability and/orperformance, further comprises at least one calcium ion and/or zinc ion.In some alternative embodiments, the composition comprising the isolatedthermolysin variants having improved stability and/or performance,further comprises at least one stabilizer. In a subset of theseembodiments, the stabilizer is chosen from borax, glycerol, zinc ions,calcium ions, and calcium formate. In some embodiments, the stabilizeris at least one competitive inhibitor that stabilizes the thermolysin inthe presence of an anionic surfactant. Alternatively, the compositionscomprising the isolated thermolysin variant having improved stabilityand/or performance, comprise at least one calcium ion and/or zinc ion,in combination with at least one stabilizer. Any one of the stabilizersrecited above may be combined with the at least one calcium ion and/orzinc ion to provide the compositions comprising at least one thermolysinvariant. In a subset of these embodiments, the stabilizer is chosen fromborax, glycerol, zinc ions, calcium ions, and calcium formate. In someother embodiments, the stabilizer is at least one competitive inhibitorthat stabilizes the thermolysin in the presence of an anionicsurfactant. In some embodiments, the thermolysin variant having improvedstability and/or performance is a Geobacillus thermolysin variant havingan amino acid sequence comprising one or more substitutions at positionschosen from positions equivalent to positions 6, 7, 49, 56, 58, 61, 63,65, 75, 128, 151, 156, 196, 273, 278, and 280 of the amino acid sequenceset forth as SEQ ID NO:3. In a subset of these embodiments, the one ormore substitutions comprise one, two, three, four or five substitutionsat positions chosen from positions equivalent to positions 6, 7, 49, 56,58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280 of the aminoacid sequence set forth as SEQ ID NO:3. In further embodiments, theinvention provides an isolated Geobacillus thermolysin variant having anamino acid sequence comprising one or more substitutions at positionschosen from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56,58, 61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261,265, 273, 278, 280 and 297 of the amino acid sequence set forth as SEQID NO:3, and having improved stability and/or performance. In a subsetof these embodiments, the one or more substitutions comprise one, two,three, four or five substitutions at positions chosen from positionsequivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85,108, 128, 129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297of the amino acid sequence set forth as SEQ ID NO:3. In some otherembodiments, the thermolysin variant having improved stability and/orperformance comprises one or more substitutions chosen from the group ofthe substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M,V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L,T049N, T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P,S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V,Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T,Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y,T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L,T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651,S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T,Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D,G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N,T278S, T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N,T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D,S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V,Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as listed in Table7-1, Table 8-1 and Table 8-2. In a subset of these embodiments, the oneor more substitutions comprise one, two, three, four or fivesubstitutions chosen from the group of substitutions T006G, T006H,T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y,V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y,T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I,A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T,Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T,I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R,T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R,A056Y, A058S, S065C, S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I,Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M,Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K,I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W,Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L,N280M, N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y,Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S,Y151T, and I156E, as listed in Table 7-1, Table 8-1 and Table 8-2.

In other embodiments, the invention provides a composition comprising anisolated thermolysin variant having improved stability and/orperformance, in combination with at least one additional enzyme orenzyme derivative chosen from proteases, amylases, lipases, mannanases,pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.In some embodiments, the composition comprising the isolated thermolysinvariant having improved stability and/or performance, and at least oneadditional enzyme or enzyme derivative chosen from proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases, further comprises at least onestabilizer. In a subset of these embodiments, the stabilizer is chosenfrom borax, glycerol, zinc ions, calcium ions, and calcium formate. Insome embodiments, the stabilizer is at least one competitive inhibitorthat stabilizes the thermolysin in the presence of an anionicsurfactant. Alternatively, the compositions comprising isolatedthermolysin variants having improved stability and/or performance,comprise at least one calcium ion and/or zinc ion, in combination withat least one stabilizer. Any one of the stabilizers recited above may becombined with the at least one calcium ion and/or zinc ion to providethe compositions comprising at least one thermolysin variant. In asubset of these embodiments, the stabilizer is chosen from borax,glycerol, zinc ions, calcium ions, and calcium formate. In some otherembodiments, the stabilizer is at least one competitive inhibitor thatstabilizes the thermolysin in the presence of an anionic surfactant. Insome embodiments, the thermolysin variant having improved stabilityand/or performance is a Geobacillus thermolysin variant having an aminoacid sequence comprising one or more substitutions at positions chosenfrom positions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75,128, 151, 156, 196, 273, 278, and 280 of the amino acid sequence setforth as SEQ ID NO:3. In a subset of these embodiments, the one or moresubstitutions comprise one, two, three, four or five substitutions atpositions chosen from positions equivalent to positions 6, 7, 49, 56,58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280 of the aminoacid sequence set forth as SEQ ID NO:3. In further embodiments, theinvention provides an isolated Geobacillus thermolysin variant having anamino acid sequence comprising one or more substitutions at positionschosen from positions equivalent to positions 4, 6, 7, 36, 49, 53, 56,58, 61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261,265, 273, 278, 280 and 297 of the amino acid sequence set forth as SEQID NO:3, and having improved stability and/or performance. In a subsetof these embodiments, the one or more substitutions comprise one, two,three, four or five substitutions at positions chosen from positionsequivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85,108, 128, 129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297of the amino acid sequence set forth as SEQ ID NO:3. In some otherembodiments, the thermolysin variant having improved stability and/orperformance comprises one or more substitutions chosen from the group ofthe substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M,V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L,T049N, T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P,S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V,Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T,Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y,T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L,T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651,S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T,Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D,G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N,T278S, T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N,T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D,S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V,Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as listed in Table7-1, Table 8-1 and Table 8-2. In a subset of these embodiments, the oneor more substitutions comprise one, two, three, four or fivesubstitutions chosen from the group of substitutions T006G, T006H,T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y,V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y,T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I,A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T,Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T,I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R,T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R,A056Y, A058S, S065C, S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I,Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M,Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K,I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W,Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L,N280M, N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y,Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S,Y151T, and I156E, as listed in Table 7-1, Table 8-1 and Table 8-2.

In some embodiments, the present invention provides cleaningcompositions comprising an isolated thermolysin variant having improvedstability and/or performance. In some embodiments, the cleaningcomposition comprising the isolated thermolysin variant having improvedstability and/or performance, further comprises at least one calcium ionand/or zinc ion. In some alternative embodiments, the cleaningcomposition comprising the isolated thermolysin variant having improvedstability and/or performance, further comprises at least one stabilizer.In a subset of these embodiments, the stabilizer is chosen from borax,glycerol, zinc ions, calcium ions, and calcium formate. In someembodiments, the stabilizer is at least one competitive inhibitor thatstabilizes the thermolysin in the presence of an anionic surfactant.Alternatively, the cleaning composition comprising the isolatedthermolysin variant having improved stability and/or performance,comprises at least one calcium ion and/or zinc ion, in combination withat least one stabilizer. Any one of the stabilizers recited above may becombined with the at least one calcium ion and/or zinc ion to providethe compositions comprising at least one thermolysin variant. In asubset of these embodiments, the stabilizer is chosen from borax,glycerol, zinc ions, calcium ions, and calcium formate. In some otherembodiments, the stabilizer is at least one competitive inhibitor thatstabilizes the thermolysin in the presence of an anionic surfactant. Insome embodiments, the thermolysin variant comprised in the cleaningcompositions and having improved stability and/or performance is aGeobacillus thermolysin variant having an amino acid sequence comprisingone or more substitutions at positions chosen from positions equivalentto positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273,278, and 280 of the amino acid sequence set forth as SEQ ID NO:3. In asubset of these embodiments, the one or more substitutions comprise one,two, three, four or five substitutions at positions chosen frompositions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128,151, 156, 196, 273, 278, and 280 of the amino acid sequence set forth asSEQ ID NO:3. In further embodiments, the thermolysin variant comprisedin the cleaning compositions and having improved stability and/orperformance is a Geobacillus thermolysin variant having an amino acidsequence comprising one or more substitutions at positions chosen frompositions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63,65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265, 273, 278,280 and 297 of the amino acid sequence set forth as SEQ ID NO:3, andhaving improved stability and/or performance. In a subset of theseembodiments, the one or more substitutions comprise one, two, three,four or five substitutions at positions chosen from positions equivalentto positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128,129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of theamino acid sequence set forth as SEQ ID NO:3. In some other embodiments,the thermolysin variant comprised in the cleaning compositions andhaving improved stability and/or performance is a Geobacillusthermolysin variant having an amino acid sequence comprising one or moresubstitutions chosen from the group of the substitutions T006G, T006H,T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y,V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y,T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I,A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T,Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T,I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R,T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R,A056Y, A058S, S065C, S065E, S0651, S065T, S065V, S065Y, Q128C, Q128I,Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M,Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K,I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W,Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L,N280M, N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y,Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S,Y151T, and I156E, as listed in Table 7-1, Table 8-1 and Table 8-2. In asubset of these embodiments, the one or more substitutions comprise one,two, three, four or five substitutions chosen from the group ofsubstitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P, T006Q,T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P,V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N,T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K,S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y,Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V,Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K,T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M,T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V,Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H,Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S,T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q,T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E,S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y,Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as listed in Table 7-1,Table 8-1 and Table 8-2.

In other embodiments, the invention provides a cleaning compositioncomprising an isolated thermolysin variant having improved stabilityand/or performance, in combination with at least one additional enzymeor enzyme derivative chosen from proteases, amylases, lipases,mannanases, pectinases, cutinases, oxidoreductases, hemicellulases, andcellulases. In some embodiments, the cleaning composition comprises theisolated thermolysin variant having improved stability and/orperformance, and at least one additional enzyme or enzyme derivativechosen from proteases, amylases, lipases, mannanases, pectinases,cutinases, oxidoreductases, hemicellulases, and cellulases furthercomprises at least one stabilizer. In a subset of these embodiments, thestabilizer is chosen from borax, glycerol, zinc ions, calcium ions, andcalcium formate. In some embodiments, the stabilizer is at least onecompetitive inhibitor that stabilizes the thermolysin in the presence ofan anionic surfactant. Alternatively, the cleaning compositionscomprising the isolated thermolysin variant having improved stabilityand/or performance, comprise at least one calcium ion and/or zinc ion,in combination with at least one stabilizer. Any one of the stabilizersrecited above may be combined with the at least one calcium ion and/orzinc ion to provide the compositions comprising at least one thermolysinvariant. In a subset of these embodiments, the stabilizer is chosen fromborax, glycerol, zinc ions, calcium ions, and calcium formate. In someother embodiments, the stabilizer is at least one competitive inhibitorthat stabilizes the thermolysin in the presence of an anionicsurfactant. In some embodiments, the thermolysin variant comprised inthe cleaning compositions and having improved stability and/orperformance is a Geobacillus thermolysin variant having an amino acidsequence comprising one or more substitutions at positions chosen frompositions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128,151, 156, 196, 273, 278, and 280 of the amino acid sequence set forth asSEQ ID NO:3. 9 In a subset of these embodiments, the one or moresubstitutions comprise one, two, three, four or five substitutions atpositions chosen from positions equivalent to positions 6, 7, 49, 56,58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280 of the aminoacid sequence set forth as SEQ ID NO:3. In further embodiments, thethermolysin variant comprised in the cleaning compositions and havingimproved stability and/or performance is a Geobacillus thermolysinvariant having an amino acid sequence comprising one or moresubstitutions at positions chosen from positions equivalent to positions4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128, 129, 151,156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of the amino acidsequence set forth as SEQ ID NO:3, and having improved stability and/orperformance. In a subset of these embodiments, the one or moresubstitutions comprise one, two, three, four or five substitutions atpositions chosen from positions equivalent to positions 4, 6, 7, 36, 49,53, 56, 58, 61, 63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196,261, 265, 273, 278, 280 and 297 of the amino acid sequence set forth asSEQ ID NO:3. In some other embodiments, the thermolysin variantcomprised in the cleaning compositions and having improved stabilityand/or performance is a Geobacillus thermolysin variant having an aminoacid sequence comprising one or more substitutions chosen from the groupof the substitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P,T006Q, T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M,V007P, V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L,T049N, T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P,S065K, S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V,Q128Y, Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T,Y151V, Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y,T278K, T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L,T049M, T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, 50651,S065T, S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A,Y151C, Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T,Y151V, Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D,G196H, Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N,T278S, T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N,T049Q, T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D,S065E, S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V,Q128Y, Y151A, Y151C, Y151N, Y151S, Y151T, and I156E, as listed in Table7-1, Table 8-1 and Table 8-2. In a subset of these embodiments, the oneor more substitutions comprise one, two, three, four or fivesubstitutions chosen from the group of substitutions T006G, T006H,T006I, T006K, T006M, T006N, T006P, T006Q, T006R, T006V, T006W, T006Y,V007F, V007H, V007K, V007L, V007M, V007P, V007Q, V007R, V007T, V007Y,T049G, T049H, T049I, T049K, T049L, T049N, T049P, T049Q, T049W, A058I,A058P, A058R, F063I, F063L, F063P, S065K, S065Y, Y075G, Y075M, Y075T,Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y, Y151D, Y151E, Y151H, Y151K,Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V, Y151W, I156M, I156R, I156T,I156W, G196R, Q273I, Q273P, Q273Y, T278K, T278M, T278P, N280K, N280R,T006A, T006C, T049D, T049I, T049L, T049M, T049N, T049S, A056C, A056R,A056Y, A058S, S065C, S065E, 50651, S065T, S065V, S065Y, Q128C, Q128I,Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151D, Y151E, Y151H, Y151M,Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V, Y151W, I156E, I156H, I156K,I156M, I156R, I156T, I156W, G196D, G196H, Q273A, Q273N, Q273T, Q273W,Q273Y, T278C, T278H, T278M, T278N, T278S, T278Y, N280E, N280I, N280L,N280M, N280S, T006C, T049D, T049N, T049Q, T049S, A056C, A056E, A058C,A058E, Q061E, Q061M, S065C, S065D, S065E, S065P, S065V, S065W, S065Y,Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C, Y151N, Y151S,Y151T, and I156E, as listed in Table 7-1, Table 8-1 and Table 8-2.

In some embodiments, any one of the cleaning compositions comprising athermolysin variant having improved stability and/or performance asrecited herein, is a detergent. In some embodiments, the compositionsare detergent compositions. In other embodiments, the compositions areliquid.

In some embodiments, the present invention provides a compositioncomprising a thermolysin variant having improved stability and/orperformance e.g. a cleaning composition, comprising at least about0.0001 weight percent of the thermolysin variant; or from about 0.001 toabout 0.5 weight percent of the same thermolysin variant. Optionally,the composition of the present invention, which comprises a thermolysinvariant having improved stability and/or performance e.g. a cleaningcomposition, further comprises at least one adjunct ingredient.Alternatively, in some other embodiments, the composition e.g. acleaning composition, further comprises a sufficient amount of a pHmodifier to provide the composition with a neat pH of from about 3 toabout 5, the composition being essentially free of materials thathydrolyze at a pH of from about pH 3 to about pH 5. In some embodiments,the materials that hydrolyze at a pH of from about pH 3 to about pH 5comprise at least one surfactant. In some preferred embodiments, thesurfactant is a sodium alkyl sulfate surfactant comprising an ethyleneoxide moiety. In some embodiments, the composition comprising athermolysin variant having improved stability and/or performance e.g. acleaning composition, is a detergent.

In addition, the present invention provides animal feed compositionscomprising an isolated thermolysin variant having improved stabilityand/or performance. In further embodiments textile processingcompositions are provided comprising an isolated thermolysin varianthaving improved stability and/or performance. In still furtherembodiments leather processing compositions are provided comprising anisolated thermolysin variant having improved stability and/orperformance.

Moreover, the present invention provides methods of cleaning, comprisingthe step of contacting a surface and/or an article comprising a fabricwith a cleaning composition comprising an isolated thermolysin varianthaving improved stability and/or performance. In some embodiments, themethods of cleaning further comprise the step of rinsing the surfaceand/or material after contacting the surface or material with thecleaning composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the amino acid sequence (SEQ ID NO:3) of the mature formof Geobacillus caldoproteolyticus thermolysin-like neutralmetalloprotease enzyme (also referred to herein as thermolysin,Proteinase-T or PrT).

FIG. 2 provides a map of the pHPLT plasmid.

FIG. 3 provides a map of the pHPLT-thermolysin expression vector.

FIG. 4A-B provides the nucleic acid sequence (SEQ ID NO:8) of thepHPLT-thermolysin expression vector.

FIG. 5 provides a graph comparing protease activity of thermolysin andNprE after incubation at room temperature in Unilever ALL Small andMighty 3× detergent.

FIG. 6 provides a graph comparing protease activity of thermolysin andNprE after incubation at room temperature in Proctor & Gamble TIDE®Fresh Breeze 1× detergent.

FIG. 7 provides a graph comparing protease activity of thermolysin andNprE after incubation at room temperature in Proctor & Gamble TIDE®Fresh Breeze 2× detergent.

FIG. 8 shows an SDS-PAGE analysis of thermolysin stability afterprolonged incubation in Unilever ALL small and mighty detergent in thepresence and absence of known metalloproteinase inhibitors.

FIG. 9 provides an alignment of the thermolysin (T) and NprE amino acidsequences. The thermolysin sequence is set forth as SEQ ID NO:3, whilethe NprE sequence is set forth as SEQ ID NO:9.

GENERAL DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions comprising atleast one thermolysin-like neutral protease enzyme with improved storagestability and/or catalytic activity. In some embodiments, thethermolysin finds use in cleaning and other applications comprisingdetergent. In some particularly preferred embodiments, the presentinvention provides methods and compositions comprising thermolysinformulated and/or engineered to resist detergent-induced inactivation.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, and recombinant DNA, which are within the skill of theart. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual,” Second Edition, ColdSpring Harbor, 1989; and Ausubel et al., “Current Protocols in MolecularBiology,” 1987). All patents, patent applications, articles andpublications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham,The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, the preferred methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the Specification as a whole.

Also, as used herein, the singular “a”, “an” and “the” includes theplural reference unless the context clearly indicates otherwise. Numericranges are inclusive of the numbers defining the range. Unless otherwiseindicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention, which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although any methodsand materials similar or equivalent to those described herein find usein the practice of the present invention, the preferred methods andmaterials are described herein. Accordingly, the terms definedimmediately below are more fully described by reference to theSpecification as a whole. Also, as used herein, the singular terms “a,”“an,” and “the” include the plural reference unless the context clearlyindicates otherwise. Unless otherwise indicated, nucleic acids arewritten left to right in 5′ to 3′ orientation; amino acid sequences arewritten left to right in amino to carboxy orientation, respectively. Itis to be understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

All documents cited are, in relevant part, incorporated herein byreference; the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

As used herein, the terms “protease,” and “proteolytic activity” referto a protein or peptide exhibiting the ability to hydrolyze peptides orsubstrates having peptide linkages. Many well known procedures exist formeasuring proteolytic activity (Kalisz, “Microbial Proteinases,” In:Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology,1988). For example, proteolytic activity may be ascertained bycomparative assays, which analyze the respective protease's ability tohydrolyze a commercial substrate. Exemplary substrates useful in suchanalysis of protease or proteolytic activity, include, but are notlimited to di-methyl casein (Sigma C-9801), bovine collagen (SigmaC-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICNBiomedical 902111). Colorimetric assays utilizing these substrates arewell known in the art (See e.g., WO 99/34011; and U.S. Pat. No.6,376,450, both of which are incorporated herein by reference. The pNAassay (See e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) alsofinds use in determining the active enzyme concentration for fractionscollected during gradient elution. This assay measures the rate at whichp-nitroaniline is released as the enzyme hydrolyzes the solublesynthetic substrate,succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(sAAPF-pNA). The rate of production of yellow color from the hydrolysisreaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration. In addition, absorbancemeasurements at 280 nm can be used to determine the total proteinconcentration. The active enzyme/total-protein ratio gives the enzymepurity.

As used herein, the terms “NprE protease,” and “NprE,” refer to theneutral metalloproteases described herein. In some preferredembodiments, the NprE protease is the protease designated herein aspurified MULTIFECT® Neutral or PMN obtained from Bacillusamyloliquefaciens. Thus, in some embodiments, the term “PMN protease”refers to a naturally occurring mature protease derived from Bacillusamyloliquefaciens. In alternative embodiments, the present inventionprovides portions of the NprE protease.

The term “Bacillus protease homologues” refers to naturally occurringproteases having substantially identical amino acid sequences to themature protease derived from Bacillus thermoproteolyticus thermolysin orpolynucleotide sequences which encode for such naturally occurringproteases, and which proteases retain the functional characteristics ofa neutral metalloprotease encoded by such nucleic acids.

As used herein, the term “thermolysin variant,” is used in reference toproteases that are similar to the wild-type thermolysin, particularly intheir function, but have mutations in their amino acid sequence thatmake them different in sequence from the wild-type protease.

As used herein, “Bacillus ssp.” refers to all of the species within thegenus “Bacillus,” which are Gram-positive bacteria classified as membersof the Family Bacillaceae, Order Bacillales, Class Bacilli. The genus“Bacillus” includes all species within the genus “Bacillus,” as known tothose of skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.It is recognized that the genus Bacillus continues to undergotaxonomical reorganization. Thus, it is intended that the genus includespecies that have been reclassified, including but not limited to suchorganisms as B. stearothermophilus, which is now named “Geobacillusstearothermophilus.” The production of resistant endospores in thepresence of oxygen is considered the defining feature of the genusBacillus, although this characteristic also applies to the recentlynamed Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus,Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus,Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, andVirgibacillus.

Related (and derivative) proteins comprise “variant proteins.” In somepreferred embodiments, variant proteins differ from a parent protein andone another by a small number of amino acid residues. The number ofdiffering amino acid residues may be one or more, preferably 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In somepreferred embodiments, the number of different amino acids betweenvariants is between 1 and 10. In some particularly preferredembodiments, related proteins and particularly variant proteins compriseat least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, arelated protein or a variant protein as used herein, refers to a proteinthat differs from another related protein or a parent protein in thenumber of prominent regions. For example, in some embodiments, variantproteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions thatdiffer from the parent protein.

Several methods are known in the art that are suitable for generatingvariants of the enzymes of the present invention, including but notlimited to site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, random mutagenesis, site-directed mutagenesis,and directed-evolution, as well as various other recombinatorialapproaches.

Characterization of wild-type and mutant proteins is accomplished viaany means or “test” suitable and is preferably based on the assessmentof properties of interest. For example, pH and/or temperature, as wellas detergent and/or oxidative stability is/are determined in someembodiments of the present invention. Indeed, it is contemplated thatenzymes having various degrees of stability in one or more of thesecharacteristics (pH, temperature, proteolytic stability, detergentstability, and/or oxidative stability) will find use.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include, but arenot limited to, a single-, double- or triple-stranded DNA, genomic DNA,cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidinebases, or other natural, chemically, biochemically modified, non-naturalor derivatized nucleotide bases. The following are non-limiting examplesof polynucleotides: genes, gene fragments, chromosomal fragments, ESTs,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. In some embodiments, polynucleotides comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracil, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. In alternative embodiments, thesequence of nucleotides is interrupted by non-nucleotide components.

As used herein, the terms “DNA construct” and “transforming DNA” areused interchangeably to refer to DNA used to introduce sequences into ahost cell or organism. The DNA may be generated in vitro by PCR or anyother suitable technique(s) known to those in the art. In particularlypreferred embodiments, the DNA construct comprises a sequence ofinterest (e.g., as an incoming sequence). In some embodiments, thesequence is operably linked to additional elements such as controlelements (e.g., promoters, etc.). The DNA construct may further comprisea selectable marker. It may further comprise an incoming sequenceflanked by homology boxes. In a further embodiment, the transforming DNAcomprises other non-homologous sequences, added to the ends (e.g.,stuffer sequences or flanks). In some embodiments, the ends of theincoming sequence are closed such that the transforming DNA forms aclosed circle. The transforming sequences may be wild-type, mutant ormodified. In some embodiments, the DNA construct comprises sequenceshomologous to the host cell chromosome. In other embodiments, the DNAconstruct comprises non-homologous sequences. Once the DNA construct isassembled in vitro it may be used to: 1) insert heterologous sequencesinto a desired target sequence of a host cell, and/or 2) mutagenize aregion of the host cell chromosome (i.e., replace an endogenous sequencewith a heterologous sequence), 3) delete target genes; and/or introducea replicating plasmid into the host.

As used herein, the terms “expression cassette” and “expression vector”refer to nucleic acid constructs generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a target cell. Therecombinant expression cassette can be incorporated into a plasmid,chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acidfragment. Typically, the recombinant expression cassette portion of anexpression vector includes, among other sequences, a nucleic acidsequence to be transcribed and a promoter. In preferred embodiments,expression vectors have the ability to incorporate and expressheterologous DNA fragments in a host cell. Many prokaryotic andeukaryotic expression vectors are commercially available. Selection ofappropriate expression vectors is within the knowledge of those of skillin the art. The term “expression cassette” is used interchangeablyherein with “DNA construct,” and their grammatical equivalents.Selection of appropriate expression vectors is within the knowledge ofthose of skill in the art.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, plasmids,cassettes and the like. In some embodiments, the polynucleotideconstruct comprises a DNA sequence encoding the protease (e.g.,precursor or mature protease) that is operably linked to a suitableprosequence (e.g., secretory, etc.) capable of effecting the expressionof the DNA in a suitable host.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orprokaryotes, or integrates into the host chromosome.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, conjugation, and transduction (See e.g.,Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, PlenumPublishing Corp., pages 57-72, 1989).

As used herein, the terms “transformed” and “stably transformed” refersto a cell that has a non-native (heterologous) polynucleotide sequenceintegrated into its genome or as an episomal plasmid that is maintainedfor at least two generations.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence, which is capable ofexpression in the host cells and where expression of the selectablemarker confers to cells containing the expressed gene the ability togrow in the presence of a corresponding selective agent or lack of anessential nutrient.

As used herein, the terms “selectable marker” and “selective marker”refer to a nucleic acid (e.g., a gene) capable of expression in hostcell, which allows for ease of selection of those hosts containing thevector. Examples of such selectable markers include but are not limitedto antimicrobials. Thus, the term “selectable marker” refers to genesthat provide an indication that a host cell has taken up an incoming DNAof interest or some other reaction has occurred.

Typically, selectable markers are genes that confer antimicrobialresistance or a metabolic advantage on the host cell to allow cellscontaining the exogenous DNA to be distinguished from cells that havenot received any exogenous sequence during the transformation. A“residing selectable marker” is one that is located on the chromosome ofthe microorganism to be transformed. A residing selectable markerencodes a gene that is different from the selectable marker on thetransforming DNA construct. Selective markers are well known to those ofskill in the art. As indicated above, preferably the marker is anantimicrobial resistant marker (e.g., amp^(R); phleo^(R); spec^(R);kan^(R); ery^(R); tee; cmp^(R); and nee (See e.g., Guerot-Fleury, Gene,167:335-337, 1995; Palmeros et al., Gene 247:255-264, 2000; andTrieu-Cuot et al., Gene, 23:331-341, 1983). Other markers useful inaccordance with the invention include, but are not limited toauxotrophic markers, such as tryptophan; and detection markers, such asβ-galactosidase.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Inpreferred embodiments, the promoter is appropriate to the host cell inwhich the target gene is being expressed. The promoter, together withother transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) is necessary to express agiven gene. In general, the transcriptional and translational regulatorysequences include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader (i.e., a signal peptide), is operably linkedto DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.

As used herein the term “gene” refers to a polynucleotide (e.g., a DNAsegment) that encodes a polypeptide and includes regions preceding andfollowing the coding regions as well as intervening sequences (introns)between individual coding segments (exons).

As used herein, “homologous genes” refers to a pair of genes fromdifferent, but usually related species, which correspond to each otherand which are identical or very similar to each other. The termencompasses genes that are separated by speciation (i.e., thedevelopment of new species) (e.g., orthologous genes), as well as genesthat have been separated by genetic duplication (e.g., paralogousgenes).

As used herein, “ortholog” and “orthologous genes” refer to genes indifferent species that have evolved from a common ancestral gene (i.e.,a homologous gene) by speciation. Typically, orthologs retain the samefunction during the course of evolution. Identification of orthologsfinds use in the reliable prediction of gene function in newly sequencedgenomes.

As used herein, “paralog” and “paralogous genes” refer to genes that arerelated by duplication within a genome. While orthologs retain the samefunction through the course of evolution, paralogs evolve new functions,even though some functions are often related to the original one.Examples of paralogous genes include, but are not limited to genesencoding trypsin, chymotrypsin, elastase, and thrombin, which are allserine proteinases and occur together within the same species.

As used herein, “homology” refers to sequence similarity or identity,with identity being preferred. This homology is determined usingstandard techniques known in the art (See e.g., Smith and Waterman, AdvAppl Math, 2:482, 1981; Needleman and Wunsch, J Mol Biol, 48:443, 1970;Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988; programs suchas GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, Madison, Wis.; and Devereux et al.,Nucl Acid Res, 12:387-395, 1984).

As used herein, an “analogous sequence” is one wherein the function ofthe gene is essentially the same as the gene based on the Geobacilluscaldoproteolyticus thermolysin. Additionally, analogous genes include atleast 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% sequence identity with the sequence of the Geobacilluscaldoproteolyticus thermolysin. In additional embodiments more than oneof the above properties applies to the sequence. Analogous sequences aredetermined by known methods of sequence alignment. A commonly usedalignment method is BLAST, although as indicated above and below, thereare other methods that also find use in aligning sequences. One exampleof a useful algorithm is PILEUP. PILEUP creates a multiple sequencealignment from a group of related sequences using progressive, pair-wisealignments. It can also plot a tree showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (Feng and Doolittle,J Mol Evol, 35:351-360, 1987). The method is similar to that describedby Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153, 1989). UsefulPILEUP parameters including a default gap weight of 3.00, a default gaplength weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedby Altschul et al., (Altschul et al., J Mol Biol, 215:403-410, 1990; andKarlin et al., Proc Natl Acad Sci USA, 90:5873-5787, 1993). Aparticularly useful BLAST program is the WU-BLAST-2 program (See,Altschul et al., Meth Enzymol, 266:460-480, 1996). WU-BLAST-2 usesseveral search parameters, most of which are set to the default values.The adjustable parameters are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSPS2 parameters are dynamic values and are established by the programitself depending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched. However, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

Thus, “percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical to the nucleotide residues of the starting sequence (i.e., thesequence of interest). A preferred method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

As used herein, the term “hybridization” refers to the process by whicha strand of nucleic acid joins with a complementary strand through basepairing, as known in the art.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm−5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° C. belowthe Tm; “intermediate stringency” at about 10-20° C. below the Tm of theprobe; and “low stringency” at about 20-25° C. below the Tm.Functionally, maximum stringency conditions may be used to identifysequences having strict identity or near-strict identity with thehybridization probe; while intermediate or low stringency hybridizationcan be used to identify or detect polynucleotide sequence homologs.

Moderate and high stringency hybridization conditions are well known inthe art. An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDSand 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C. An example of moderate stringentconditions include an overnight incubation at 37° C. in a solutioncomprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. Those of skill in theart know how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention. “Recombination,” “recombining,” and generating a“recombined” nucleic acid are generally the assembly of two or morenucleic acid fragments wherein the assembly gives rise to a chimericgene.

In a preferred embodiment, mutant DNA sequences are generated with sitesaturation mutagenesis in at least one codon. In another preferredembodiment, site saturation mutagenesis is performed for two or morecodons. In a further embodiment, mutant DNA sequences have more than50%, more than 55%, more than 60%, more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 98% homology with the wild-type sequence. In alternativeembodiments, mutant DNA is generated in vivo using any known mutagenicprocedure such as, for example, radiation, nitrosoguanidine and thelike. The desired DNA sequence is then isolated and used in the methodsprovided herein.

As used herein, the term “target sequence” refers to a DNA sequence inthe host cell that encodes the sequence where it is desired for theincoming sequence to be inserted into the host cell genome. In someembodiments, the target sequence encodes a functional wild-type gene oroperon, while in other embodiments the target sequence encodes afunctional mutant gene or operon, or a non-functional gene or operon.

As used herein, a “flanking sequence” refers to any sequence that iseither upstream or downstream of the sequence being discussed (e.g., forgenes A-B-C, gene B is flanked by the A and C gene sequences). In apreferred embodiment, the incoming sequence is flanked by a homology boxon each side. In another embodiment, the incoming sequence and thehomology boxes comprise a unit that is flanked by stuffer sequence oneach side. In some embodiments, a flanking sequence is present on only asingle side (either 3′ or 5′), but in preferred embodiments, it is oneach side of the sequence being flanked. In some embodiments, a flankingsequence is present on only a single side (either 3′ or 5′), while inpreferred embodiments it is present on each side of the sequence beingflanked.

As used herein, the term “stuffer sequence” refers to any extra DNA thatflanks homology boxes (typically vector sequences). However, the termencompasses any non-homologous DNA sequence. Not to be limited by anytheory, a stuffer sequence provides a noncritical target for a cell toinitiate DNA uptake.

As used herein, the terms “amplification” and “gene amplification” referto a process by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhibitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “co-amplification” refers to the introductioninto a single cell of an amplifiable marker in conjunction with othergene sequences (i.e., comprising one or more non-selectable genes suchas those contained within an expression vector) and the application ofappropriate selective pressure such that the cell amplifies both theamplifiable marker and the other, non-selectable gene sequences. Theamplifiable marker may be physically linked to the other gene sequencesor alternatively two separate pieces of DNA, one containing theamplifiable marker and the other containing the non-selectable marker,may be introduced into the same cell.

As used herein, the terms “amplifiable marker,” “amplifiable gene,” and“amplification vector” refer to a gene or a vector encoding a gene,which permits the amplification of that gene under appropriate growthconditions.

“Template specificity” is achieved in most amplification techniques bythe choice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (See e.g., Kacian et al., Proc Natl Acad Sci USA 69:3038,1972) and other nucleic acids are not replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (See,Chamberlin et al., Nature 228:227, 1970). In the case of T4 DNA ligase,the enzyme will not ligate the two oligonucleotides or polynucleotides,where there is a mismatch between the oligonucleotide or polynucleotidesubstrate and the template at the ligation junction (See, Wu andWallace, Genomics 4:560, 1989). Finally, Taq and Pfu polymerases, byvirtue of their ability to function at high temperature, are found todisplay high specificity for the sequences bounded and thus defined bythe primers; the high temperature results in thermodynamic conditionsthat favor primer hybridization with the target sequences and nothybridization with non-target sequences.

As used herein, the term “amplifiable nucleic acid” refers to nucleicacids, which may be amplified by any amplification method. It iscontemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample, which is analyzed for the presence of“target” (defined below). In contrast, “background template” is used inreference to nucleic acid other than sample template, which may or maynot be present in a sample. Background template is most ofteninadvertent. It may be the result of carryover, or it may be due to thepresence of nucleic acid contaminants sought to be purified away fromthe sample. For example, nucleic acids from organisms other than thoseto be detected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188,hereby incorporated by reference, which include methods for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “RT-PCR” refers to the replication andamplification of RNA sequences. In this method, reverse transcription iscoupled to PCR, most often using a one enzyme procedure in which athermostable polymerase is employed, as described in U.S. Pat. No.5,322,770, herein incorporated by reference. In RT-PCR, the RNA templateis converted to cDNA due to the reverse transcriptase activity of thepolymerase, and then amplified using the polymerizing activity of thepolymerase (i.e., as in other PCR methods).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A “restriction site” refers to a nucleotide sequence recognized andcleaved by a given restriction endonuclease and is frequently the sitefor insertion of DNA fragments. In certain embodiments of the inventionrestriction sites are engineered into the selective marker and into 5′and 3′ ends of the DNA construct.

As used herein, the term “chromosomal integration” refers to the processwhereby an incoming sequence is introduced into the chromosome of a hostcell. The homologous regions of the transforming DNA align withhomologous regions of the chromosome. Subsequently, the sequence betweenthe homology boxes is replaced by the incoming sequence in a doublecrossover (i.e., homologous recombination). In some embodiments of thepresent invention, homologous sections of an inactivating chromosomalsegment of a DNA construct align with the flanking homologous regions ofthe indigenous chromosomal region of the Bacillus chromosome.Subsequently, the indigenous chromosomal region is deleted by the DNAconstruct in a double crossover (i.e., homologous recombination).

“Homologous recombination” means the exchange of DNA fragments betweentwo DNA molecules or paired chromosomes at the site of identical ornearly identical nucleotide sequences. In a preferred embodiment,chromosomal integration is homologous recombination.

“Homologous sequences” as used herein means a nucleic acid orpolypeptide sequence having 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 88%, 85%, 80%, 75%, or 70% sequence identity to anothernucleic acid or polypeptide sequence when optimally aligned forcomparison. In some embodiments, homologous sequences have between 85%and 100% sequence identity, while in other embodiments there is between90% and 100% sequence identity, and in more preferred embodiments, thereis 95% and 100% sequence identity.

As used herein “amino acid” refers to peptide or protein sequences orportions thereof. The terms “protein,” “peptide,” and “polypeptide” areused interchangeably.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in the host cell. Examples ofheterologous proteins include enzymes such as hydrolases includingproteases. In some embodiments, the gene encoding the proteins arenaturally occurring genes, while in other embodiments, mutated and/orsynthetic genes are used.

As used herein, “homologous protein” refers to a protein or polypeptidenative or naturally occurring in a cell. In preferred embodiments, thecell is a Gram-positive cell, while in particularly preferredembodiments the cell is a Bacillus host cell. In alternativeembodiments, the homologous protein is a native protein produced byother organisms, including but not limited to E. coli, Streptomyces,Trichoderma, and Aspergillus. The invention encompasses host cellsproducing the homologous protein via recombinant DNA technology.

As used herein, an “operon region” comprises a group of contiguous genesthat are transcribed as a single transcription unit from a commonpromoter, and are thereby subject to co-regulation. In some embodiments,the operon includes a regulator gene. In most preferred embodiments,operons that are highly expressed as measured by RNA levels, but have anunknown or unnecessary function are used.

As used herein, an “antimicrobial region” is a region containing atleast one gene that encodes an antimicrobial protein.

A polynucleotide is said to “encode” an RNA or a polypeptide if, in itsnative state or when manipulated by methods known to those of skill inthe art, it can be transcribed and/or translated to produce the RNA, thepolypeptide or a fragment thereof. The anti-sense strand of such anucleic acid is also said to encode the sequences.

As is known in the art, a DNA can be transcribed by an RNA polymerase toproduce RNA, but an RNA can be reverse transcribed by reversetranscriptase to produce a DNA. Thus a DNA can encode a RNA and viceversa.

The term “regulatory segment” or “regulatory sequence” or “expressioncontrol sequence” refers to a polynucleotide sequence of DNA that isoperatively linked with a polynucleotide sequence of DNA that encodesthe amino acid sequence of a polypeptide chain to effect the expressionof the encoded amino acid sequence. The regulatory sequence can inhibit,repress, or promote the expression of the operably linked polynucleotidesequence encoding the amino acid.

“Host strain” or “host cell” refers to a suitable host for an expressionvector comprising DNA according to the present invention.

An enzyme is “overexpressed” in a host cell if the enzyme is expressedin the cell at a higher level that the level at which it is expressed ina corresponding wild-type cell.

The terms “protein” and “polypeptide” are used interchangeabilityherein. The 3-letter code for amino acids as defined in conformity withthe IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) isused through out this disclosure. It is also understood that apolypeptide may be coded for by more than one nucleotide sequence due tothe degeneracy of the genetic code.

A “prosequence” is an amino acid sequence between the signal sequenceand mature protease that is necessary for the secretion of the protease.Cleavage of the pro sequence will result in a mature active protease.

The term “signal sequence” or “signal peptide” refers to any sequence ofnucleotides and/or amino acids that participate in the secretion of themature or precursor forms of the protein. This definition of signalsequence is a functional one, meant to include all those amino acidsequences encoded by the N-terminal portion of the protein gene, whichparticipate in the effectuation of the secretion of protein. They areoften, but not universally, bound to the N-terminal portion of a proteinor to the N-terminal portion of a precursor protein. The signal sequencemay be endogenous or exogenous. The signal sequence may be that normallyassociated with the protein (e.g., protease), or may be from a geneencoding another secreted protein. One exemplary exogenous signalsequence comprises the first seven amino acid residues of the signalsequence from Bacillus subtilis subtilisin fused to the remainder of thesignal sequence of the subtilisin from Bacillus lentus (ATCC 21536).

The term “hybrid signal sequence” refers to signal sequences in whichpart of sequence is obtained from the expression host fused to thesignal sequence of the gene to be expressed. In some embodiments,synthetic sequences are utilized.

The term “mature” form of a protein or peptide refers to the finalfunctional form of the protein or peptide. To exemplify, a mature formof thermolysin includes the amino acid sequence of SEQ ID NO:3.

The term “precursor” form of a protein or peptide refers to a matureform of the protein having a prosequence operably linked to the amino orcarbonyl terminus of the protein. The precursor may also have a “signal”sequence operably linked, to the amino terminus of the prosequence. Theprecursor may also have additional polynucleotides that are involved inpost-translational activity (e.g., polynucleotides cleaved therefrom toleave the mature form of a protein or peptide).

“Naturally occurring enzyme” refers to an enzyme having the unmodifiedamino acid sequence identical to that found in nature. Naturallyoccurring enzymes include native enzymes, those enzymes naturallyexpressed or found in the particular microorganism.

The terms “derived from” and “obtained from” refer to not only aprotease produced or producible by a strain of the organism in question,but also a protease encoded by a DNA sequence isolated from such strainand produced in a host organism containing such DNA sequence.Additionally, the term refers to a protease that is encoded by a DNAsequence of synthetic and/or cDNA origin and which has the identifyingcharacteristics of the protease in question. To exemplify, “proteasesderived from Bacillus sp.” refers to those enzymes having proteolyticactivity which are naturally-produced by Bacillus sp., as well as toneutral metalloproteases like those produced by Bacillus sp. sources butwhich through the use of genetic engineering techniques are produced bynon-Geobacillus caldoproteolyticus organisms transformed with a nucleicacid encoding said neutral metalloproteases.

A “derivative” within the scope of this definition generally retains thecharacteristic proteolytic activity observed in the wild-type, native orparent form to the extent that the derivative is useful for similarpurposes as the wild-type, native or parent form. Functional derivativesof neutral metalloprotease encompass naturally occurring, syntheticallyor recombinantly produced peptides or peptide fragments having thegeneral characteristics of the neutral metalloprotease of the presentinvention.

The term “functional derivative” refers to a derivative of a nucleicacid having the functional characteristics of a nucleic acid encoding aneutral metalloprotease. Functional derivatives of a nucleic acid, whichencode neutral metalloprotease of the present invention encompassnaturally occurring, synthetically or recombinantly produced nucleicacids or fragments and encode neutral metalloprotease characteristic ofthe present invention. Wild type nucleic acid encoding neutralmetalloprotease according to the invention include naturally occurringalleles and homologues based on the degeneracy of the genetic code knownin the art.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences that are the samewhen aligned for maximum correspondence, as measured using one of thefollowing sequence comparison or analysis algorithms.

The term “optimal alignment” refers to the alignment giving the highestpercent identity score.

“Percent sequence identity,” “percent amino acid sequence identity,”“percent gene sequence identity,” and/or “percent nucleicacid/polynucloetide sequence identity,” with respect to two amino acid,polynucleotide and/or gene sequences (as appropriate), refer to thepercentage of residues that are identical in the two sequences when thesequences are optimally aligned. Thus, 80% amino acid sequence identitymeans that 80% of the amino acids in two optimally aligned polypeptidesequences are identical.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides thus refers to a polynucleotide or polypeptide thatcomprising at least 70% sequence identity, preferably at least 75%,preferably at least 80%, preferably at least 85%, preferably at least90%, preferably at least 95%, preferably at least 97%, preferably atleast 98% and preferably at least 99% sequence identity as compared to areference sequence using the programs or algorithms (e.g., BLAST, ALIGN,CLUSTAL) using standard parameters. One indication that two polypeptidesare substantially identical is that the first polypeptide isimmunologically cross-reactive with the second polypeptide. Typically,polypeptides that differ by conservative amino acid substitutions areimmunologically cross-reactive. Thus, a polypeptide is substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by a conservative substitution. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules hybridize to each other under stringent conditions (e.g.,within a range of medium to high stringency).

The term “isolated” or “purified” refers to a material that is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, the material is said to be “purified”when it is present in a particular composition in a higher or lowerconcentration than exists in a naturally occurring or wild type organismor in combination with components not normally present upon expressionfrom a naturally occurring or wild type organism. For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Such polynucleotides could be part of a vector,and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. In preferred embodiments, a nucleicacid or protein is said to be purified, for example, if it gives rise toessentially one band in an electrophoretic gel or blot.

The term “isolated”, when used in reference to a DNA sequence, refers toa DNA sequence that has been removed from its natural genetic milieu andis thus free of other extraneous or unwanted coding sequences, and is ina form suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (See e.g., Dynan and Tijan, Nature 316:774-78, 1985).The term “an isolated DNA sequence” is alternatively referred to as “acloned DNA sequence”.

The term “isolated,” when used in reference to a protein, refers to aprotein that is found in a condition other than its native environment.In a preferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins. An isolated protein ismore than 10% pure, preferably more than 20% pure, and even morepreferably more than 30% pure, as determined by SDS-PAGE. Furtheraspects of the invention encompass the protein in a highly purified form(i.e., more than 40% pure, more than 60% pure, more than 80% pure, morethan 90% pure, more than 95% pure, more than 97% pure, and even morethan 99% pure), as determined by SDS-PAGE.

The following cassette mutagenesis method may be used to facilitate theconstruction of the enzyme variants of the present invention, althoughother methods may be used. First, as described herein, anaturally-occurring gene encoding the enzyme is obtained and sequencedin whole or in part. Then, the sequence is scanned for a point at whichit is desired to make a mutation (deletion, insertion or substitution)of one or more amino acids in the encoded enzyme. The sequences flankingthis point are evaluated for the presence of restriction sites forreplacing a short segment of the gene with an oligonucleotide pool whichwhen expressed will encode various mutants. Such restriction sites arepreferably unique sites within the protein gene so as to facilitate thereplacement of the gene segment. However, any convenient restrictionsite that is not overly redundant in the enzyme gene may be used,provided the gene fragments generated by restriction digestion can bereassembled in proper sequence. If restriction sites are not present atlocations within a convenient distance from the selected point (from 10to 15 nucleotides), such sites are generated by substituting nucleotidesin the gene in such a fashion that neither the reading frame nor theamino acids encoded are changed in the final construction. Mutation ofthe gene in order to change its sequence to conform to the desiredsequence is accomplished by M13 primer extension in accord withgenerally known methods. The task of locating suitable flanking regionsand evaluating the needed changes to arrive at two convenientrestriction site sequences is made routine by the redundancy of thegenetic code, a restriction enzyme map of the gene and the large numberof different restriction enzymes. Note that if a convenient flankingrestriction site is available, the above method need be used only inconnection with the flanking region that does not contain a site.

Once the naturally-occurring DNA and/or synthetic DNA is cloned, therestriction sites flanking the positions to be mutated are digested withthe cognate restriction enzymes and a plurality of endtermini-complementary oligonucleotide cassettes are ligated into thegene. The mutagenesis is simplified by this method because all of theoligonucleotides can be synthesized so as to have the same restrictionsites, and no synthetic linkers are necessary to create the restrictionsites.

As used herein, “corresponding to,” refers to a residue at theenumerated position in a protein or peptide, or a residue that isanalogous, homologous, or equivalent to an enumerated residue in aprotein or peptide.

As used herein, “corresponding region,” generally refers to an analogousposition along related proteins or a parent protein.

As used herein, the term, “combinatorial mutagenesis” refers to methodsin which libraries of variants of a starting sequence are generated. Inthese libraries, the variants contain one or several mutations chosenfrom a predefined set of mutations. In addition, the methods providemeans to introduce random mutations, which were not members of thepredefined set of mutations. In some embodiments, the methods includethose set forth in U.S. application Ser. No. 09/699,250, filed Oct. 26,2000, hereby incorporated by reference. In alternative embodiments,combinatorial mutagenesis methods encompass commercially available kits(e.g., QUIKCHANGE® Multisite, Stratagene, San Diego, Calif.).

As used herein, the term “library of mutants” refers to a population ofcells which are identical in most of their genome but include differenthomologues of one or more genes. Such libraries can be used, forexample, to identify genes or operons with improved traits.

As used herein, the terms “starting gene” and “parent gene” refer to agene of interest that encodes a protein of interest that is to beimproved and/or changed using the present invention.

As used herein, the terms “multiple sequence alignment” and “MSA” referto the sequences of multiple homologs of a starting gene that arealigned using an algorithm (e.g., Clustal W).

As used herein, the terms “consensus sequence” and “canonical sequence”refer to an archetypical amino acid sequence against which all variantsof a particular protein or sequence of interest are compared. The termsalso refer to a sequence that sets forth the nucleotides that are mostoften present in a DNA sequence of interest. For each position of agene, the consensus sequence gives the amino acid that is most abundantin that position in the MSA.

As used herein, the term “consensus mutation” refers to a difference inthe sequence of a starting gene and a consensus sequence. Consensusmutations are identified by comparing the sequences of the starting geneand the consensus sequence obtained from a MSA. In some embodiments,consensus mutations are introduced into the starting gene such that itbecomes more similar to the consensus sequence. Consensus mutations alsoinclude amino acid changes that change an amino acid in a starting geneto an amino acid that is more frequently found in an MSA at thatposition relative to the frequency of that amino acid in the startinggene. Thus, the term consensus mutation comprises all single amino acidchanges that replace an amino acid of the starting gene with an aminoacid that is more abundant than the amino acid in the MSA.

The terms “modified sequence” and “modified genes” are usedinterchangeably herein to refer to a sequence that includes a deletion,insertion or interruption of naturally occurring nucleic acid sequence.In some preferred embodiments, the expression product of the modifiedsequence is a truncated protein (e.g., if the modification is a deletionor interruption of the sequence). In some particularly preferredembodiments, the truncated protein retains biological activity. Inalternative embodiments, the expression product of the modified sequenceis an elongated protein (e.g., modifications comprising an insertioninto the nucleic acid sequence). In some embodiments, an insertion leadsto a truncated protein (e.g., when the insertion results in theformation of a stop codon). Thus, an insertion may result in either atruncated protein or an elongated protein as an expression product.

As used herein, the terms “mutant sequence” and “mutant gene” are usedinterchangeably and refer to a sequence that has an alteration in atleast one codon occurring in a host cell's wild-type sequence. Theexpression product of the mutant sequence is a protein with an alteredamino acid sequence relative to the wild-type. The expression productmay have an altered functional capacity (e.g., enhanced enzymaticactivity).

The terms “mutagenic primer” or “mutagenic oligonucleotide” (usedinterchangeably herein) are intended to refer to oligonucleotidecompositions which correspond to a portion of the template sequence andwhich are capable of hybridizing thereto. With respect to mutagenicprimers, the primer will not precisely match the template nucleic acid,the mismatch or mismatches in the primer being used to introduce thedesired mutation into the nucleic acid library. As used herein,“non-mutagenic primer” or “non-mutagenic oligonucleotide” refers tooligonucleotide compositions that match precisely to the templatenucleic acid. In one embodiment of the invention, only mutagenic primersare used. In another preferred embodiment of the invention, the primersare designed so that for at least one region at which a mutagenic primerhas been included, there is also non-mutagenic primer included in theoligonucleotide mixture. By adding a mixture of mutagenic primers andnon-mutagenic primers corresponding to at least one of the mutagenicprimers, it is possible to produce a resulting nucleic acid library inwhich a variety of combinatorial mutational patterns are presented. Forexample, if it is desired that some of the members of the mutant nucleicacid library retain their parent sequence at certain positions whileother members are mutant at such sites, the non-mutagenic primersprovide the ability to obtain a specific level of non-mutant memberswithin the nucleic acid library for a given residue. The methods of theinvention employ mutagenic and non-mutagenic oligonucleotides which aregenerally between 10-50 bases in length, more preferably about 15-45bases in length. However, it may be necessary to use primers that areeither shorter than 10 bases or longer than 50 bases to obtain themutagenesis result desired. With respect to corresponding mutagenic andnon-mutagenic primers, it is not necessary that the correspondingoligonucleotides be of identical length, but only that there is overlapin the region corresponding to the mutation to be added.

Primers may be added in a pre-defined ratio according to the presentinvention. For example, if it is desired that the resulting library havea significant level of a certain specific mutation and a lesser amountof a different mutation at the same or different site, by adjusting theamount of primer added, it is possible to produce the desired biasedlibrary. Alternatively, by adding lesser or greater amounts ofnon-mutagenic primers, it is possible to adjust the frequency with whichthe corresponding mutation(s) are produced in the mutant nucleic acidlibrary.

The terms “wild-type sequence,” or “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein-engineering project. The wild-type sequence may encode eithera homologous or heterologous protein. A homologous protein is one thehost cell would produce without intervention. A heterologous protein isone that the host cell would not produce but for the intervention.

As used herein, the term “equivalent” when used in reference to theposition of an amino acid residue in a thermolysin protein refers to theposition of an amino acid residue in a thermolysin variant thatcorresponds in position in the primary sequence of the unmodifiedprecursor e.g. wild-type thermolysin. In order to establish the positionof equivalent amino acid positions in a thermolysin, the amino acidsequence of the thermolysin that is modified to generate the thermolysinvariant is directly compared to the thermolysin of SEQ ID NO:3. Afteraligning the residues, allowing for insertions and deletions in order tomaintain alignment (i.e. avoiding the elimination of conserved residuesthrough arbitrary deletion or insertion), the residues at positionsequivalent to particular amino acid positions in the sequence of thethermolysin of SEQ ID NO:3 are defined.

The term “oxidation stable” refers to proteases of the present inventionthat retain a specified amount of enzymatic activity over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposed toor contacted with bleaching agents or oxidizing agents. In someembodiments, the proteases retain at least 50%, 60%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 96%, 97%, 98% or 99% proteolytic activity after contactwith a bleaching or oxidizing agent over a given time period, forexample, at least 1 minute, 3 minutes, 5 minutes, 8 minutes, 12 minutes,16 minutes, 20 minutes, etc.

The term “chelator stable” refers to proteases of the present inventionthat retain a specified amount of enzymatic activity over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposed toor contacted with chelating agents. In some embodiments, the proteasesretain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%,98% or 99% proteolytic activity after contact with a chelating agentover a given time period, for example, at least 10 minutes, 20 minutes,40 minutes, 60 minutes, 100 minutes, etc.

The terms “thermally stable” and “thermostable” refer to proteases ofthe present invention that retain a specified amount of enzymaticactivity after exposure to identified temperatures over a given periodof time under conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention, for example while exposedaltered temperatures. Altered temperatures include increased ordecreased temperatures. In some embodiments, the proteases retain atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%proteolytic activity after exposure to altered temperatures over a giventime period, for example, at least 60 minutes, 120 minutes, 180 minutes,240 minutes, 300 minutes, etc.

As used herein, the term “chemical stability” refers to the stability ofa protein (e.g., an enzyme) towards chemicals that adversely affect itsactivity. In some embodiments, such chemicals include, but are notlimited to hydrogen peroxide, peracids, anionic detergents, cationicdetergents, non-ionic detergents, chelants, etc. However, it is notintended that the present invention be limited to any particularchemical stability level nor range of chemical stability. In particular,the terms “detergent stable” and “LAS stable” refer to proteases of thepresent invention that retain a specified amount of enzymatic activityafter exposure to a detergent composition over a given period of timeunder conditions prevailing during the proteolytic, hydrolyzing,cleaning or other process of the invention. In some embodiments, theproteases retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%,96%, 97%, 98% or 99% proteolytic activity after exposure to detergentover a given time period, for example, at least 60 minutes, 120 minutes,180 minutes, 240 minutes, 300 minutes, etc.

The term “enhanced stability” in the context of an oxidation, chelator,thermal and/or pH stable protease refers to a higher retainedproteolytic activity over time as compared to other neutralmetalloproteases and/or wild-type enzymes.

The term “diminished stability” in the context of an oxidation,chelator, thermal and/or pH stable protease refers to a lower retainedproteolytic activity over time as compared to other neutralmetalloproteases and/or wild-type enzymes.

As used herein, the term “cleaning composition” includes, unlessotherwise indicated, granular or powder-form all-purpose or “heavy-duty”washing agents, especially cleaning detergents; liquid, gel orpaste-form all-purpose washing agents, especially the so-calledheavy-duty liquid types; liquid fine-fabric detergents; hand dishwashingagents or light duty dishwashing agents, especially those of thehigh-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

Unless otherwise noted, all component or composition levels are inreference to the active level of that component or composition, and areexclusive of impurities, for example, residual solvents or by-products,which may be present in commercially available sources.

Enzyme components weights are based on total active protein. Allpercentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated.

The term “cleaning activity” refers to the cleaning performance achievedby the protease under conditions prevailing during the proteolytic,hydrolyzing, cleaning or other process of the invention. In someembodiments, cleaning performance is determined by the application ofvarious cleaning assays concerning enzyme sensitive stains, for examplegrass, blood, milk, or egg protein as determined by variouschromatographic, spectrophotometric or other quantitative methodologiesafter subjection of the stains to standard wash conditions. Exemplaryassays include, but are not limited to those described in WO 99/34011,and U.S. Pat. No. 6,605,458 (both of which are herein incorporated byreference), as well as those methods included in the Examples.

The term “cleaning effective amount” of a protease refers to thequantity of protease described hereinbefore that achieves a desiredlevel of enzymatic activity in a specific cleaning composition. Sucheffective amounts are readily ascertained by one of ordinary skill inthe art and are based on many factors, such as the particular proteaseused, the cleaning application, the specific composition of the cleaningcomposition, and whether a liquid or dry (e.g., granular, bar)composition is required, etc.

The term “cleaning adjunct materials” as used herein, means any liquid,solid or gaseous material selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, granule,powder, bar, paste, spray, tablet, gel; or foam composition), whichmaterials are also preferably compatible with the protease enzyme usedin the composition. In some embodiments, granular compositions are in“compact” form, while in other embodiments, the liquid compositions arein a “concentrated” form.

As used herein, a “low detergent concentration” system includesdetergents where less than about 800 ppm of detergent components arepresent in the wash water. Japanese detergents are typically consideredlow detergent concentration systems, as they have usually haveapproximately 667 ppm of detergent components present in the wash water.

As used herein, a “medium detergent concentration” systems includesdetergents wherein between about 800 ppm and about 2000 ppm of detergentcomponents are present in the wash water. North American detergents aregenerally considered to be medium detergent concentration systems asthey have usually approximately 975 ppm of detergent components presentin the wash water. Brazilian detergents typically have approximately1500 ppm of detergent components present in the wash water.

As used herein, “high detergent concentration” systems includesdetergents wherein greater than about 2000 ppm of detergent componentsare present in the wash water. European detergents are generallyconsidered to be high detergent concentration systems as they haveapproximately 3000-8000 ppm of detergent components in the wash water.

As used herein, “fabric cleaning compositions” include hand and machinelaundry detergent compositions including laundry additive compositionsand compositions suitable for use in the soaking and/or pretreatment ofstained fabrics (e.g., clothes, linens, and other textile materials).

As used herein, “non-fabric cleaning compositions” include non-textile(i.e., fabric) surface cleaning compositions, including but not limitedto dishwashing detergent compositions, oral cleaning compositions,denture cleaning compositions, and personal cleansing compositions.

The “compact” form of the cleaning compositions herein is best reflectedby density and, in terms of composition, by the amount of inorganicfiller salt. Inorganic filler salts are conventional ingredients ofdetergent compositions in powder form. In conventional detergentcompositions, the filler salts are present in substantial amounts,typically 17-35% by weight of the total composition. In contrast, incompact compositions, the filler salt is present in amounts notexceeding 15% of the total composition. In some embodiments, the fillersalt is present in amounts that do not exceed 10%, or more preferably,5%, by weight of the composition. In some embodiments, the inorganicfiller salts are selected from the alkali and alkaline-earth-metal saltsof sulfates and chlorides. A preferred filler salt is sodium sulfate.

DETAILED DESCRIPTION OF THE INVENTION

Neutral metalloendopeptidases (i.e., neutral metalloproteases) (EC3.4.24.4) belong to a protease class that has an absolute requirementfor zinc ions for catalytic activity. These enzymes are optimally activeat neutral pH and are in the 30 to 40 kDa size range. Neutralmetalloproteases bind between two and four calcium ions that contributeto the structural stability of the protein. The bound metal ion at theactive site of metalloproteases is an essential feature that allows theactivation of a water molecule. The water molecule then functions as thenucleophile and cleaves the carbonyl group of the peptide bond.

The neutral zinc-binding metalloprotease family includes the bacterialenzyme thermolysin, and thermolysin-like proteases (TLPs), as well ascarboxypeptidase A (a digestive enzyme), and the matrix metalloproteasesthat catalyze the reactions in tissue remodeling and degradation. Theonly well characterized of these proteases, with respect to stabilityand function is thermolysin, which hydrolyzes protein bonds on theamino-terminal side of hydrophobic amino acid residues. Thermolysin is athermostable neutral zinc metalloproteinase first identified in theculture broth of Bacillus thermoproteolyticus Rokko. Subsequently, asimilar neutral metalloprotease was identified in Geobacilluscaldoprotelyticus, and this enzyme is also referred to herein asthermolysin. Natural and engineered proteases, such as thermolysin areoften expressed in Bacillus subtilis (O'Donohue et al., Biochem J,300:599-603, 1994), and several have been applied in detergentformulations to remove proteinaceous stains. Today, thermolysin is usedin industry, especially for the enzymatic synthesis of N-carbobenzoxy1-Asp-1-Phe methyl ester, a precursor of the artificial sweeteneraspartame.

In general however, the serine proteases have been more widely utilizedin detergents, at least partially due to the relative ease with whichthese proteases can be stabilized.

Indeed, metalloproteases are less frequently used in industry, andparticularly in the detergent industry for a number of reasons. Theseenzymes involve more complex protein systems, as the enzymes have theabsolute requirement for calcium and zinc ions for stability andfunction, respectively. Further, the detergent solution as well as thewater used in the laundry process often contains components thatinterfere with the binding of the ions by the enzyme, or chelate theseions, resulting in a decrease or loss of proteolytic function anddestabilization of the protease.

Detailed Description of Cleaning and Detergent Formulations of thePresent Invention

Unless otherwise noted, all component or composition levels providedherein are made in reference to the active level of that component orcomposition, and are exclusive of impurities, for example, residualsolvents or by-products, which may be present in commercially availablesources. Enzyme components weights are based on total active protein.All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated.

In the exemplified detergent compositions, the enzymes levels areexpressed by pure enzyme by weight of the total composition and unlessotherwise specified, the detergent ingredients are expressed by weightof the total compositions.

Cleaning Compositions Comprising Neutral Metalloprotease

The neutral metalloproteases of the present invention are useful informulating various detergent compositions. The cleaning composition ofthe present invention may be advantageously employed for example, inlaundry applications, hard surface cleaning, automatic dishwashingapplications, as well as cosmetic applications such as dentures, teeth,hair and skin. However, due to the unique advantages of increasedeffectiveness in lower temperature solutions and the superiorcolor-safety profile, the enzymes of the present invention are ideallysuited for laundry applications such as the bleaching of fabrics.Furthermore, the enzymes of the present invention find use in bothgranular and liquid compositions.

The enzymes of the present invention also find use in cleaning additiveproducts. A cleaning additive product including at least one enzyme ofthe present invention is ideally suited for inclusion in a wash processwhen additional bleaching effectiveness is desired. Such instancesinclude, but are not limited to low temperature solution cleaningapplications. The additive product may be, in its simplest form, one ormore neutral metalloprotease enzyme as provided by the presentinvention. In some embodiments, the additive is packaged in dosage formfor addition to a cleaning process where a source of peroxygen isemployed and increased bleaching effectiveness is desired. In someembodiments, the single dosage form comprises a pill, tablet, gelcap orother single dosage unit including pre-measured powders and/or liquids.In some embodiments, filler and/or carrier material(s) are included, inorder to increase the volume of such composition. Suitable filler orcarrier materials include, but are not limited to, various salts ofsulfate, carbonate and silicate as well as talc, clay and the like. Insome embodiments filler and/or carrier materials for liquid compositionsinclude water and/or low molecular weight primary and secondary alcoholsincluding polyols and diols. Examples of such alcohols include, but arenot limited to, methanol, ethanol, propanol and isopropanol. In someembodiments, the compositions comprise from about 5% to about 90% ofsuch materials. In additional embodiments, acidic fillers are used toreduce the pH of the composition. In some alternative embodiments thecleaning additive includes at least one activated peroxygen source asdescribed below and/or adjunct ingredients as more fully describedbelow.

The cleaning compositions and cleaning additives of the presentinvention require an effective amount of neutral metalloprotease enzymeas provided in the present invention. In some embodiments, the requiredlevel of enzyme is achieved by the addition of one or more species ofneutral metalloprotease provided by the present invention. Typically,the cleaning compositions of the present invention comprise at least0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 toabout 0.5, or even from about 0.01 to about 0.1 weight percent of atleast one neutral metalloprotease provided by the present invention.

In some preferred embodiments, the cleaning compositions provided hereinare typically formulated such that, during use in aqueous cleaningoperations, the wash water has a pH of from about 5.0 to about 11.5, orin alternative embodiments, even from about 6.0 to about 10.5. In somepreferred embodiments, liquid product formulations are typicallyformulated to have a neat pH from about 3.0 to about 9.0, while in somealternative embodiments the formulation has a neat pH from about 3 toabout 5. In some preferred embodiments, granular laundry products aretypically formulated to have a pH from about 8 to about 11. Techniquesfor controlling pH at recommended usage levels include the use ofbuffers, alkalis, acids, etc., and are well known to those skilled inthe art.

In some particularly preferred embodiments, when at least one neutralmetalloprotease is employed in a granular composition or liquid, theneutral metalloprotease is in the form of an encapsulated particle toprotect the enzyme from other components of the granular compositionduring storage. In addition, encapsulation also provides a means ofcontrolling the availability of the neutral metalloprotease(s) duringthe cleaning process and may enhance performance of the neutralmetalloprotease(s). It is contemplated that the encapsulated neutralmetalloproteases of the present invention will find use in varioussettings. It is also intended that the neutral metalloprotease beencapsulated using any suitable encapsulating material(s) and method(s)known in the art.

In some preferred embodiments, the encapsulating material typicallyencapsulates at least part of the neutral metalloprotease catalyst. Insome embodiments, the encapsulating material is water-soluble and/orwater-dispersible. In some additional embodiments, the encapsulatingmaterial has a glass transition temperature (Tg) of 0° C. or higher (Seee.g., WO 97/11151, particularly from page 6, line 25 to page 7, line 2,for more information regarding glass transition temperatures).

In some embodiments, the encapsulating material is chosen fromcarbohydrates, natural or synthetic gums, chitin and chitosan, celluloseand cellulose derivatives, silicates, phosphates, borates, polyvinylalcohol, polyethylene glycol, paraffin waxes and combinations thereof.In some embodiments in which the encapsulating material is acarbohydrate, it is chosen from monosaccharides, oligosaccharides,polysaccharides, and combinations thereof. In some preferredembodiments, the encapsulating material is a starch (See e.g., EP 0 922499; U.S. Pat. No. 4,977,252. U.S. Pat. No. 5,354,559, and U.S. Pat. No.5,935,826, for descriptions of some exemplary starches).

In additional embodiments, the encapsulating material comprises amicrosphere made from plastic (e.g., thermoplastics, acrylonitrile,methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixturesthereof; commercially available microspheres that find use include, butare not limited to EXPANCEL® [Casco Products, Stockholm, Sweden], PM6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, and Q-CEL® [PQ Corp.,Valley Forge, Pa.], LUXSIL® and SPHERICEL1® [Potters Industries, Inc.,Carlstadt, N.J. and Valley Forge, Pa.]).

Processes of Making and Using of Applicants' Cleaning Composition

In some preferred embodiments compositions of the present invention areformulated into any suitable form and prepared by any process chosen bythe formulator, (See e.g., U.S. 5,879,584, U.S. Pat. No. 5,691,297, U.S.Pat. No. 5,574,005, U.S. Pat. No. 5,569,645, U.S. Pat. No. 5,565,422,U.S. Pat. No. 5,516,448, U.S. Pat. No. 5,489,392, and U.S. Pat. No.5,486,303, for some non-limiting examples). In some embodiments in whicha low pH cleaning composition is desired, the pH of such composition isadjusted via the addition of an acidic material such as HCl.

Adjunct Materials

While not essential for the purposes of the present invention, in someembodiments, the non-limiting list of adjuncts described herein aresuitable for use in the cleaning compositions of the present invention.Indeed, in some embodiments, adjuncts are incorporated into the cleaningcompositions of the present invention. In some embodiments, adjunctmaterials assist and/or enhance cleaning performance, treat thesubstrate to be cleaned, and/or modify the aesthetics of the cleaningcomposition (e.g., perfumes, colorants, dyes, etc.). It is understoodthat such adjuncts are in addition to the neutral metalloproteases ofthe present invention. The precise nature of these additionalcomponents, and levels of incorporation thereof, depends on the physicalform of the composition and the nature of the cleaning operation forwhich it is to be used. Suitable adjunct materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, deposition aids, dispersants, additional enzymes, andenzyme stabilizers, catalytic materials, bleach activators, bleachboosters, hydrogen peroxide, sources of hydrogen peroxide, preformedperacids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,perfumes, structure elasticizing agents, fabric softeners, carriers,hydrotropes, processing aids and/or pigments. In addition to thoseprovided explicitly herein, additional examples are known in the art(See e.g., U.S. Pat. Nos. 5,576,282, 6,306,812 B1 and 6,326,348 B1). Insome embodiments, the aforementioned adjunct ingredients constitute thebalance of the cleaning compositions of the present invention.

Surfactants—

In some embodiments, the cleaning compositions of the present inventioncomprise at least one surfactant or surfactant system, wherein thesurfactant is chosen from nonionic surfactants, anionic surfactants,cationic surfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants, and mixtures thereof. In some low pHcleaning composition embodiments (e.g., compositions having a neat pH offrom about 3 to about 5), the composition typically does not containalkyl ethoxylated sulfate, as it is believed that such surfactant may behydrolyzed in acidic compositions.

In some embodiments, the surfactant is present at a level of from about0.1% to about 60%, while in alternative embodiments, the level is fromabout 1% to about 50%, while in still further embodiments, the level isfrom about 5% to about 40%, by weight of the cleaning composition.

Builders—

In some embodiments, the cleaning compositions of the present inventioncomprise one or more detergent builders or builder systems. In someembodiments incorporating at least one builder, the cleaningcompositions comprise at least about 1%, from about 3% to about 60% oreven from about 5% to about 40% builder by weight of the cleaningcomposition.

Builders include, but are not limited to, the alkali metal, ammonium andalkanolammonium salts of polyphosphates, alkali metal silicates,alkaline earth and alkali metal carbonates, aluminosilicate builderspolycarboxylate compounds. ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof. Indeed, it is contemplated that any suitablebuilder will find use in various embodiments of the present invention.

Chelating Agents—

In some embodiments, the cleaning compositions of the present inventioncontain at least one chelating agent. Suitable chelating agents include,but are not limited to copper, iron and/or manganese chelating agentsand mixtures thereof. In embodiments in which at least one chelatingagent is used, the cleaning compositions of the present inventioncomprise from about 0.1% to about 15% or even from about 3.0% to about10% chelating agent by weight of the subject cleaning composition.

Deposition Aid—

In some embodiments, the cleaning compositions of the present inventioninclude at least one deposition aid. Suitable deposition aids include,but are not limited to polyethylene glycol, polypropylene glycol,polycarboxylate, soil release polymers such as polytelephthalic acid,clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite,halloysite, and mixtures thereof.

Dye Transfer Inhibiting Agents—

In some embodiments, the cleaning compositions of the present inventioninclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof.

In embodiments in which at least one dye transfer inhibiting agent isused, the cleaning compositions of the present invention comprise fromabout 0.0001% to about 10%, from about 0.01% to about 5%, or even fromabout 0.1% to about 3% by weight of the cleaning composition.

Dispersants—

In some embodiments, the cleaning compositions of the present inventioncontains at least one dispersants. Suitable water-soluble organicmaterials include, but are not limited to the homo- or co-polymericacids or their salts, in which the polycarboxylic acid comprises atleast two carboxyl radicals separated from each other by not more thantwo carbon atoms.

Enzymes—

In some embodiments, the cleaning compositions of the present inventioncomprise one or more detergent enzymes, which provide cleaningperformance and/or fabric care benefits. Examples of suitable enzymesinclude, but are not limited to, hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, keratinases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,malanases, B-glucanases, arabinosidases, hyaluronidase, chondroitinase,laccase, and amylases, or mixtures thereof. In some embodiments, acombination of enzymes is used (i.e., a “cocktail”) comprisingconventional applicable enzymes like protease, lipase, cutinase and/orcellulase in conjunction with amylase is used.

Enzyme Stabilizers—

In some embodiments of the present invention, the enzymes used in thedetergent formulations of the present invention are stabilized. It iscontemplated that various techniques for enzyme stabilization will finduse in the present invention. For example, in some embodiments, theenzymes employed herein are stabilized by the presence of water-solublesources of zinc (II), calcium (II) and/or magnesium (II) ions in thefinished compositions that provide such ions to the enzymes, as well as.other metal ions (e.g., barium (II), scandium (II), iron (II), manganese(II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II),and oxovanadium (IV)).

Catalytic Metal Complexes—

In some embodiments, the cleaning compositions of the present inventioncontain one or more catalytic metal complexes. In some embodiments, ametal-containing bleach catalyst finds use. In some preferredembodiments, the metal bleach catalyst comprises a catalyst systemcomprising a transition metal cation of defined bleach catalyticactivity, (e.g., copper, iron, titanium, ruthenium, tungsten,molybdenum, or manganese cations), an auxiliary metal cation havinglittle or no bleach catalytic activity (e.g., zinc or aluminum cations),and a sequestrate having defined stability constants for the catalyticand auxiliary metal cations, particularly ethylenediaminetetraaceticacid, ethylenediaminetetra (methylenephosphonic acid) and water-solublesalts thereof are used (See e.g., U.S. Pat. No. 4,430,243).

In some embodiments, the cleaning compositions of the present inventionare catalyzed by means of a manganese compound. Such compounds andlevels of use are well known in the art (See e.g., U.S. Pat. No.5,576,282).

In additional embodiments, cobalt bleach catalysts find use in thecleaning compositions of the present invention. Various cobalt bleachcatalysts are known in the art (See e.g., U.S. Pat. No. 5,597,936, andU.S. Pat. No. 5,595,967). Such cobalt catalysts are readily prepared byknown procedures (See e.g., U.S. Pat. No. 5,597,936, and U.S. Pat. No.5,595,967).

In additional embodiments, the cleaning compositions of the presentinvention include a transition metal complex of a macropolycyclic rigidligand (“MRL”). As a practical matter, and not by way of limitation, insome embodiments, the compositions and cleaning processes provided bythe present invention are adjusted to provide on the order of at leastone part per hundred million of the active MRL species in the aqueouswashing medium, and in some preferred embodiments, provide from about0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of theMRL in the wash liquor.

Preferred transition-metals in the instant transition-metal bleachcatalyst include, but are not limited to manganese, iron and chromium.Preferred MRLs also include, but are not limited to special ultra-rigidligands that are cross-bridged (e.g.,5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitabletransition metal MRLs are readily prepared by known procedures (Seee.g., WO 00/32601, and U.S. Pat. No. 6,225,464).

Processes of Making and Using Cleaning Compositions

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any suitable process chosen by theformulator, (See e.g., U.S. Pat. No. 5,879,584, U.S. Pat. No. 5,691,297,U.S. Pat. No. 5,574,005, U.S. Pat. No. 5,569,645, U.S. Pat. No.5,565,422, U.S. Pat. No. 5,516,448, U.S. Pat. No. 5,489,392, U.S. Pat.No. 5,486,303, U.S. Pat. No. 4,515,705, U.S. Pat. No. 4,537,706, U.S.Pat. No. 4,515,707, U.S. Pat. No. 4,550,862, U.S. Pat. No. 4,561,998,U.S. Pat. No. 4,597,898, U.S. Pat. No. 4,968,451, U.S. Pat. No.5,565,145, U.S. Pat. No. 5,929,022, U.S. Pat. No. 6,294,514, and U.S.Pat. No. 6,376,445, all of which are incorporated herein by referencefor some non-limiting examples).

Method of Use

In preferred embodiments, the cleaning compositions of the presentinvention find use in cleaning surfaces and/or fabrics. In someembodiments, at least a portion of the surface and/or fabric iscontacted with at least one embodiment of the cleaning compositions ofthe present invention, in neat form or diluted in a wash liquor, andthen the surface and/or fabric is optionally washed and/or rinsed. Forpurposes of the present invention, “washing” includes, but is notlimited to, scrubbing, and mechanical agitation. In some embodiments,the fabric comprises any fabric capable of being laundered in normalconsumer use conditions. In preferred embodiments, the cleaningcompositions of the present invention are used at concentrations of fromabout 500 ppm to about 15,000 ppm in solution. In some embodiments inwhich the wash solvent is water, the water temperature typically rangesfrom about 5° C. to about 90° C. In some preferred embodiments forfabric cleaning, the water to fabric mass ratio is typically from about1:1 to about 30:1.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: ° C. (degrees Centigrade); rpm (revolutions perminute); H₂O (water); HCl (hydrochloric acid); aa and AA (amino acid);bp (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg andug (micrograms); mg (milligrams); ng (nanograms); μl and ul(microliters); ml (milliliters); mm (millimeters); nm (nanometers); μmand um (micrometer); M (molar); mM (millimolar); μM and uM (micromolar);U (units); V (volts); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); OD₂₈₀ (optical density at 280 nm); OD₄₀₅ (opticaldensity at 405 nm); OD₆₀₀ (optical density at 600 nm); PAGE(polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphatebuffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]);LAS (lauryl sodium sulfonate); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); TAED(N,N,N′N′-tetraacetylethylenediamine); BES (polyesstersulfone); MES(2-morpholinoethanesulfonic acid, monohydrate; f.w. 195.24; Sigma #M-3671); CaCl₂ (calcium chloride, anhydrous; f.w. 110.99; Sigma #C-4901); DMF (N,N-dimethylformamide, f.w. 73.09, d=0.95); Abz-AGLA-Nba(2-aminobenzoyl-L-alanyl-glycyl-L-leucyl-L-alanino-4-nitrobenzylamide,f.w. 583.65; Bachem # H-6675, VWR catalog #100040-598); SBG1% (SuperBroth with Glucose; 6 g Soytone [Difco], 3 g yeast extract, 6 g NaCl, 6g glucose); the pH was adjusted to 7.1 with NaOH prior to sterilizationusing methods known in the art; w/v (weight to volume); v/v (volume tovolume); SEQUEST® (SEQUEST database search program, University ofWashington); MS (mass spectroscopy); BMI (blood, milk, ink); SRI (StainRemoval Index); Npr and npr (neutral metalloprotease gene); Npr and npr(neutral metalloprotease enzyme); NprE and nprE (B. amyloliquefaciensneutral metalloprotease); PrT and prt (proteinase-T enzyme); and TLP(thermolysin-like protease).

The following abbreviations apply to companies whose products orservices may have been referred to in the experimental examples: TIGR(The Institute for Genomic Research, Rockville, Md.); AATCC (AmericanAssociation of Textile and Coloring Chemists); Amersham (Amersham LifeScience, Inc. Arlington Heights, Ill.); Corning (Corning International,Corning, N.Y.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.);Pierce (Pierce Biotechnology, Rockford, Ill.); Equest (Equest, WarwickInternational Group, Inc., Flintshire, UK); EMPA (EidgenossischeMaterial Prufungs and Versuch Anstalt, St. Gallen, Switzerland); CFT(Center for Test Materials, Vlaardingen, The Netherlands); Amicon(Amicon, Inc., Beverly, Mass.); ATCC (American Type Culture Collection,Manassas, Va.); Becton Dickinson (Becton Dickinson Labware, LincolnPark, N.J.); Perkin-Elmer (Perkin-Elmer, Wellesley, Mass.); Rainin(Rainin Instrument, LLC, Woburn, Mass.); Eppendorf (Eppendorf AG,Hamburg, Germany); Waters (Waters, Inc., Milford, Mass.); PerseptiveBiosystems (Perseptive Biosystems, Ramsey, Minn.); Molecular Probes(Molecular Probes, Eugene, Oreg.); BioRad (BioRad, Richmond, Calif.);Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Cargill (Cargill,Inc., Minneapolis, Minn.); Difco (Difco Laboratories, Detroit, Mich.);GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); NewBrunswick (New Brunswick Scientific Company, Inc., Edison, N.J.);Thermoelectron (Thermoelectron Corp., Waltham, Mass.); BMG (BMG Labtech,GmbH, Offenburg, Germany); Greiner (Greiner Bio-One, Kremsmuenster,Austria); Novagen (Novagen, Inc., Madison, Wis.); Novex (Novex, SanDiego, Calif.); Finnzymes (Finnzymes OY, Finland) Qiagen (Qiagen, Inc.,Valencia, Calif.); Invitrogen (Invitrogen Corp., Carlsbad, Calif.);Sigma (Sigma Chemical Co., St. Louis, Mo.); DuPont Instruments(Asheville, N.Y.); Global Medical Instrumentation or GMI (Global MedicalInstrumentation; Ramsey, Minn.); MJ Research (MJ Research, Waltham,Mass.); Infors (Infors AG, Bottmingen, Switzerland); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); Roche (Hoffmann LaRoche, Inc., Nutley, N.J.); Agilent (Agilent Technologies, Palo Alto,Calif.); S-Matrix (S-Matrix Corp., Eureka, Calif.); US Testing (UnitedStates Testing Co., Hoboken, N.Y.); West Coast Analytical Services (WestCoast Analytical Services, Inc., Santa Fe Springs, Calif.); Ion BeamAnalysis Laboratory (Ion Bean Analysis Laboratory, The University ofSurrey Ion Beam Centre (Guildford, UK); BaChem (BaChem AG, Bubendorf,Switzerland); Molecular Devices (Molecular Devices, Inc., Sunnyvale,Calif.); MicroCal (Microcal, Inc., Northhampton, Mass.); ChemicalComputing (Chemical Computing Corp., Montreal, Canada); NCBI (NationalCenter for Biotechnology Information, Bethesda, Md.); Argo Bioanalytica(Argo Bioanalytica. Inc, New Jersey); Vydac (Grace Vydac, Hesperia,Calif.); Minolta (Konica Minolta, Ramsey, N.J.); Zeiss (Carl Zeiss,Inc., Thornwood, N.Y.); Sloning BioTechnology GmbH (Puchheim, Germany);and Procter and Gamble (Cincinnati, Ohio).

Example 1 Assays

The following assays were used in the examples described below. Anydeviations from the protocols provided below are indicated in theexamples. In these experiments, a spectrophotometer was used to measurethe absorbance of the products formed after the completion of thereactions.

A. Bradford Assay for Protein Content Determination in 96-Well Plates

The Bradford Dye reagent (Quick Start) assay was used to determine theprotein concentration in thermolysin samples on a microtiter plate (MTP)scale.

In this assay system, the chemical and reagent solutions used were:

-   -   Bradford Quick Start Dye Reagent™ (BIO-RAD Catalogue No.        500-0205)    -   Dilution Buffer (10 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN®-80)

The equipment used was a Biomek FX Robot (Beckman Coulter) and aSpectraMAX MTP Reader (type 340; Molecular Devices). MTPs were obtainedfrom Costar (type 9017).

In the test, 200 μl Bradford Dye Reagent was pipetted into each well,followed by the addition of 15 μl dilution buffer. Finally, 10 μl of thethermolysin containing filtered culture supernatants was added to thewells. After thorough mixing, the MTPs were incubated for at least 10minutes at room temperature. Possible air bubbles were blown away andthe absorbance of the wells was read at 595 nm.

To determine the protein concentration, the background reading (i.e.,from uninoculated wells) was subtracted from the sample readings. Theresulting OD₅₉₅ values provided a relative measure of the proteincontent in the samples. The Bradford results were linear with respect tothermolysin protein concentrations between 10 to 100 μg protein per ml.

B. Microswatch Assay for Testing Protease Performance

The stain removal performance of thermolysin and variants thereof wasdetermined using microswatches (EMPA 116) on a MTP scale. Thermolysincontaining protease samples were obtained from filtered broth ofcultures grown in microtiter plates for 3 days at 37° C. with shaking at280 rpm under humidified aeration.

In this assay system, the chemical and reagent solutions used were:

-   -   Thermolysin containing culture supernatants (˜100-200 μg protein        per ml)    -   TIDE® 2× (nil enzymes) detergent (P&G)    -   Dilution Buffer (10 mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN®-80)

The equipment used was a Biomek FX Robot (Beckman Coulter), a SpectraMAXMTP Reader (type 340; Molecular Devices), and an iEMS incubator/shaker(Thermo/Labsystems). MTPs were obtained from Costar (type 9017).

TIDE® 2× Liquid Detergent Preparation (US Conditions):

Milli-Q water was adjusted to 6 gpg water hardness using a (Ca/Mg 3:1)hardness stock solution (282.3 g/L CaCl₂.2H₂0, 130.1 g/L MgCl₂.6H₂O),0.78 g/l detergent TIDE® 2× was added, and the detergent solution wasstirred vigorously for at least 15 minutes. Then, 5 mM HEPES was addedand the pH adjusted to 8.2.

Microswatches:

Microswatches of ¼ inch circular diameter were obtained from CFT(Vlaardingen, The Netherlands). Before cutting the swatches, the fabric(EMPA 116) was pre-washed in de-ionised water for 20 minutes at ambienttemperature, and subsequently air-dried.

Two microswatches were placed vertically into each well of a 96-wellmicrotiter plate to expose the whole surface area (i.e., not flat on thebottom of the well).

Test Method:

The incubator was set to 20° C. The filtered culture broth samples weretested at an appropriate concentration by dilution with a mixture of 10mM NaCl, 0.1 mM CaCl₂, 0.005% TWEEN®-80 solution. The detergent solutionwas prepared as described above. Then, 190 μl of detergent solution wereadded to each well of the MTP, containing microswatches. To thismixture, 10 μl of diluted enzyme solution were added to each well (toprovide a total volume of 200 μl/well). The MTP was covered with a plateseal and placed in an incubator for 30 minutes at 20° C., with agitationat 1400 rpm (iEMS incubator). Following incubation under the appropriateconditions, 100 μl of solution from each well was removed and placedinto a new MTP. Subsequently this MTP, containing 100 μl ofsolution/well, was read at 405 nm in a MTP-Reader. Blank controls,containing 2 microswatches/well and detergent, without the addition ofthermolysin containing samples, were also included in the test.

Calculation of the BMI (Blood/Milk/Ink) Performance:

The observed absorbance value was corrected for the blank value(obtained after incubation of microswatches in the absence of addedenzyme). The resulting absorbance was a measure for the hydrolyticactivity. For each sample (thermolysin or a variant) the performanceindex (PI) was calculated. The performance index is a comparison of theperformance of the variant (actual value) and the standard thermolysinenzyme (theoretical value) at the same protein concentration. Inaddition, the theoretical values were calculated, using the parametersof the Langmuir equation of the standard enzyme.

A performance index greater than 1 (PI>1) identified a better variant(as compared to the standard [e.g., wild-type]), while a PI of 1 (PI=1)identified a variant that performs the same as the standard, and a PIless than 1 (PI<1) identified a variant that performs worse than thestandard. Thus, the PI identified winners, as well as variants that areless desirable for use under certain circumstances.

C. Stability Assay in the Presence of Detergent

The stability of thermolysin and variants thereof was measured afterincubation under defined conditions in the presence of 25% TIDE® 2×detergent. The initial and residual activity was determined.

In this assay system, the chemical and reagent solutions used were:

-   -   thermolysin containing culture supernatants (˜100-200 μg protein        per ml)    -   TIDE® 2× liquid detergent with and without DTPA chelator (P&G)    -   27.5% TIDE® 2× detergent solution with DTPA in 5.5 mM HEPES        buffer, pH 8.2 (TIDE®+ solution)    -   27.5% TIDE® 2× detergent solution w/o DTPA in 5.5 mM HEPES        buffer, pH 8.2 (TIDE®− solution)    -   MES assay buffer (55.5 mM MES/NaOH, 2.6 mM CaCl₂, 0.005%        TWEEN®-80, pH 6.5)

The equipment used was a Biomek FX Robot (Beckman Coulter), afluorescence spectrophotometer (FLUOstar Optima; BMG), an iEMSincubator/shaker (Thermo/Labsystems). MTPs were obtained from Costar(type 9017) and from Greiner (black plate, type 655076).

Test Method Unstressed Conditions:

First, 20 μl thermolysin containing culture supernatant was diluted with180 μl MES assay buffer. Then, 20 μl diluted supernatant was dilutedfurther with 180 μl MES assay buffer. Subsequently 10 μl of thisdilution was transferred into 190 μl AGLA-substrate solution in apre-warmed plate (Greiner 655076) at 25° C. Any air bubbles present wereblown away and the plate was measured according to the AGLA proteaseassay protocol described below.

Stressed Conditions:

First, 20 μl of culture supernatant was diluted with 180 μl 27.5% TIDE®+detergent solution and placed in the iEMS shaker. The plate covered witha plate seal was incubated for a total of 60 minutes at 32° C. at 900rpm. In addition, 20 μl of culture supernatant was diluted with 180 μl27.5% TIDE®− solution and placed in the iEMS shaker. This plate coveredwith a plate seal was incubated for a total of 180 minutes at 50° C. at900 rpm.

Subsequently after the respective incubations, 20 μl of either of thesesolutions were diluted with 180 μl MES assay buffer and 10 μl of thisdilution were diluted with 190 μl AGLA-substrate solution in apre-warmed plate (Greiner 655076) at 25° C.

Any air bubbles present were blown away and the plate was measuredaccording to the AGLA protease assay protocol described below.

Calculations of TIDE® 2× Stability

Fluorescence measurements were taken at excitation of 350 nm andemission of 420 nm. The spectrofluorometer software calculated thereaction rates (=slope) of the increase in fluorescence for each well toa linearly regressed line of (milli-) RFU/min. The ratio of the residualand initial AGLA activity was used to express the 25% TIDE® 2× stabilityas follows:

Percentage of residual activity=[slope of stressed]*100/[slope ofunstressed]

For each sample (thermolysin and variants thereof) the performance indexwas calculated by dividing the residual activity of the variant by theresidual activity of thermolysin. The performance index compared thestability of the variant and the standard thermolysin enzyme (e.g., wildtype or parental enzyme), determined under the same conditions.

A performance index (PI) greater than 1 (PI>1) identified a bettervariant (as compared to the standard [e.g., wild-type]), while a PI of 1(PI=1) identified a variant that displayed the same stability as thestandard, and a PI less than 1 (PI<1) identified a variant that was lessstable as compared to the standard. Thus, the PI identified winners, aswell as variants that are less desirable for use under certaincircumstances.

D.2-Aminobenzoyl-L-alanyl-L-glycyl-L-leucyl-L-alanino-4-nitrobenzylamide(Abz-AGLA-Nba) Protease Assay

The method described herein provides a degree of technical detail thatyields reproducible protease assay data independent of time and place.While the assay can be adapted to a given laboratory condition, any dataobtained through a modified procedure must be reconciled with resultsproduced by the original method. Neutral metallo-proteases cleave thepeptide bond between glycyl- and leucyl- of2-Aminobenzoyl-L-alanyl-L-glycyl-L-leucyl-L-alanino-4-nitrobenzylamide(Abz-AGLA-Nba). Free 2-Aminobenzoyl-L-alanylglycine (Abz-AG) in solutionhas a fluorescence emission maximum at 415 nm with an excitation maximumof 340 nm. Fluorescence of Abz-AG is quenched by nitrobenzylamide in theintact Abz-AGLA-Nba molecule.

In these experiments, the liberation of Abz-AG by protease cleavage ofAbz-AGLA-Nba was monitored by fluorescence spectrometry (Ex. 350/Em.420). The rate of appearance of Abz-AG was a measure of proteolyticactivity.

In this assay system, the chemical and reagent solutions used were:

-   -   MES substrate buffer—52.5 mM MES, 2.5 mM CaCl₂, 0.005%        TWEEN®-80, pH 6.5    -   MES assay buffer—55.5 mM MES, 2.6 mM CaCl₂, 0.005% TWEEN®-80, pH        6.5    -   Abz-AGLA-Nba stock solution—48 mM Abz-AGLA-Nba in        dimethylformamid (28.2 mg/ml DMF)

The equipment used was a Biomek FX Robot (Beckman Coulter), aspectrofluorometer (FLUOstar Optima; BMG), an iEMS incubator/shaker(Thermo/Labsystems) and Innova incubator (Innova-4230; New Brunswick).MTPs were obtained from Costar (type 9017) and from Greiner (blackplate, type 655076).

Test Method

The Abz-AGLA-Nba assay solution was prepared by adding 1 ml of theAbz-AGLA-Nba stock to 19 ml MES substrate buffer and mixed well for atleast 2 minutes. Subsequently the thermolysin or variants thereofcontaining culture supernatants were diluted with MES assay buffer to aconcentration of 1-6 μg protein per ml.

The assay was performed by adding 10 μl of diluted protease solution toeach well, followed by the addition of 190 μl Abz-AGLA-Nba assaysolution that was pre-equilibrated for at least 15 minutes at 25° C. Thesolutions were vigorously mixed, and the liberation of Abz-AG byprotease cleavage of Abz-AGLA-Nba was monitored by fluorescencespectrometry at 25° C. in kinetic mode with excitation set at 350 nm andemission set at 420 nm. The rate of appearance of Abz-AG was a measureof proteolytic activity in the samples. The protease activity wasexpressed as RFU (relative fluorescence units·min⁻¹).

Example 2 Thermolysin Production in B. subtilis

In this Example, experiments conducted to produce thermolysin in B.subtilis are described. The full-length thermolysin of Geobacilluscaldoproteolyticus is greater than 99% identical to the thermolysinprecursor of Bacillus thermoproteolyticus Rokko, and to the bacillolysin(NprS) precursor of Bacillus stearothermophilus. As such the terms“thermolysin,” “bacillolysin,” “proteinase-T” and “PrT” are usedinterchangeably herein to refer to the neutral metalloprotease enzyme ofG. caldoproteolyticus. The DNA sequence (thermolysin leader, thermolysinpro and thermolysin mature from Geobacillus caldoproteolyticus) providedbelow, encodes the thermolysin precursor protein:

(SEQ ID NO: 1) ATGAAAATGAAAATGAAATTAGCATCGTTTGGTCTTGCAGCAGGACTAGCGGCCCAAGTATTTTTACCTTACAATGCGCTGGCTTCAACGGAACACGTTACATGGAACCAACAATTTCAAACCCCTCAATTCATCTCCGGTGATCTGCTGAAAGTGAATGGCACATCCCCAGAAGAACTCGTCTATCAATATGTTGAAAAAAACGAAAACAAGTTTAAATTTCATGAAAACGCTAAGGATACTCTACAATTGAAAGAAAAGAAAAATGATAACCTTGGTTTTACGTTTATGCGCTTCCAACAAACGTATAAAGGGATTCCTGTGTTTGGAGCAGTAGTAACTGCGCACGTGAAAGATGGCACGCTGACGGCGCTATCAGGGACACTGATTCCGAATTTGGACACGAAAGGATCCTTAAAAAGCGGGAAGAAATTGAGTGAGAAACAAGCGCGTGACATTGCTGAAAAAGATTTAGTGGCAAATGTAACAAAGGAAGTACCGGAATATGAACAGGGAAAAGACACCGAGTTTGTTGTTTATGTCAATGGGGACGAGGCTTCTTTAGCGTACGTTGTCAATTTAAACTTTTTAACTCCTGAACCAGGAAACTGGCTGTATATCATTGATGCCGTAGACGGAAAAATTTTAAATAAATTTAACCAACTTGACGCCGCAAAACCAGGTGATGTGAAGTCG ATAACAGGAACATCAACTGTCGGAGTGGGAAGAGGAGTACTTGGTGATCAAAAAAATATTAATACAACCTACTCTACGTACTACTATTTACAAGATAATACGCGTGGAAATGGGATTTTCACGTATGATGCGAAATACCGTACGACATTGCCGGGAAGCTTATGGGCAGATGCAGATAACCAATTTTTTGCGAGCTATGATGCTCCAGCGGTTGATGCTCATTATTACGCTGGTGTGACATATGACTACTATAAAAATGTTCATAACCGTCTCAGTTACGACGGAAATAATGCAGCTATTAGATCATCCGTTCATTATAGCCAAGGCTATAATAACGCATTTTGGAACGGTTCGCAAATGGTGTATGGCGATGGTGATGGTCAAACATTTATTCCACTTTCTGGTGGTATTGATGTGGTCGCACATGAGTTAACGCATGCGGTAACCGATTATACAGCCGGACTCATTTATCAAAACGAATCTGGTGCAATTAATGAGGCAATATCTGATATTTTTGGAACGTTAGTCGAATTTTACGCTAACAAAAATCCAGATTGGGAAATTGGAGAGGATGTGTATACACCTGGTATTTCAGGGGATTCGCTCCGTTCGATGTCCGATCCGGCAAAGTATGGTGATCCAGATCACTATTCAAAGCGCTATACAGGCACGCAAGATAATGGCGGGGTTCATATCAATAGCGGAATTATCAACAAAGCCGCTTATTTGATTAGCCAAGGCGGTACGCATTACGGTGTGAGTGTTGTCGGAATCGGACGCGATAAATTGGGGAAAATTTTCTATCGTGCATTAACGCAATATTTAACACCAACGTCCAACTTTAGCCAACTTCGTGCTGCCGCTGTTCAATCAGCCACTGACTTGTACGGTTCGACAAGCCAGGAAGTCGCTTCTGTGAAGCAGGCCTTTGATGCGGTAGGGGTGAAAT AA

In the above sequence, bold indicates the DNA encoding the maturethermolysin protease, standard font indicates the DNA encoding theleader sequence (thermolysin leader), and underlined text indicates DNAencoding the pro sequence (thermolysin pro). The amino acid sequence(thermolysin leader, thermolysin pro and thermolysin mature DNAsequence) provided below (SEQ ID NO:2), corresponds to the full lengththermolysin precursor protein. In this sequence, underlined indicatesthe pro sequence and bold indicates the mature thermolysin protease.

(SEQ ID NO: 2) MKMKMKLASFGLAAGLAAQVFLPYNALASTEHVTWNQQFQTPQFISGDLLKVNGTSPEELVYQYVEKNENKFKFHENAKDTLQLKEKKNDNLGFTFMRFQQTYKGIPVFGAVVTAHVKDGTLTALSGTLIPNLDTKGSLKSGKKLSEKQARDIAEKDLVANVTKEVPEYEQGKDTEFVVYVNGDEASLAYVVNLNFLTPEPGNWLYIIDAVDGKILNKFNQLDAAKPGDVKS ITGTSTVGVGRGVLGDQKNINTTYSTYYYLQDNTRGNGIFTYDAKYRTTLPGSLWADADNQFFASYDAPAVDAHYYAGVTYDYYKNVHNRLSYDGNNAAIRSSVHYSQGYNNAFWNGSQMVYGDGDGQTFIPLSGGIDVVAHELTHAVTDYTAGLIYQNESGAINEAISDIFGTLVEFYANKNPDWEIGEDVYTPGISGDSLRSMSDPAKYGDPDHYSKRYTGTQDNGGVHINSGIINKAAYLISQGGTHYGVSVVGIGRDKLGKIFYRALTQYLTPTSNFSQLRAAAVQSATDLYGSTSQEVASVKQAFDAVGVKThe mature thermolysin sequence is set forth as SEQ ID NO:3 and shown inFIG. 1. This sequence was used as the basis for making the variantlibraries describe herein.

(SEQ ID NO: 3) ITGTSTVGVGRGVLGDQKNINTTYSTYYYLQDNTRGNGIFTYDAKYRTTLPGSLWADADNQFFASYDAPAVDAHYYAGVTYDYYKNVHNRLSYDGNNAAIRSSVHYSQGYNNAFWNGSQMVYGDGDGQTFIPLSGGIDVVAHELTHAVTDYTAGLIYQNESGAINEAISDIFGTLVEFYANKNPDWEIGEDVYTPGISGDSLRSMSDPAKYGDPDHYSKRYTGTQDNGGVHINSGIINKAAYLISQGGTHYGVSVVGIGRDKLGKIFYRALTQYLTPTSNFSQLRAAAVQSATDLYGSTSQEVASVKQAFDAVGVK

The pHPLT-thermolysin expression vector was constructed by amplifyingthe thermolysin gene from genomic DNA of Geobacillus caldoproteolyticus(Chen et al., Extremophiles, 8:489-498, 2004) and from pHPLT plasmid DNA(van Solingen et al., Extremophiles, 5:333-341, 2001). A map for thepHPLT plasmid is provided in FIG. 2. This plasmid contains thethermostable amylase LAT promoter (P_(LAT)) of Bacillus licheniformis todrive expression of thermolysin. The thermolysin gene was amplified fromthe genomic DNA using Finnzymes (Finnzymes OY, Espoo, Finland) PhusionHigh-Fidelity DNA Polymerase (Catalog No. F-530L) and the followingprimers:

pHPLT-ProT-FW: (SEQ ID NO: 4) GAGAGGGTAAAGAATGAAAATGAAAATGAAATTAGCATCproT-EcoRI-RV: (SEQ ID NO: 5)GTTAACCTGCAGGAATTCTTATTTCACCCCTACCGCATCAAAGGCC

The pHPLT fragment was amplified from the plasmid pHPLT using FinnzymesPhusion High-Fidelity DNA Polymerase and the following primers:

pHPLT-ProT-RV: (SEQ ID NO: 6) CATTTTCATTTTCATTCTTTACCCTCTCCTTTTGCTAGACproT-EcoRI-FW: (SEQ ID NO: 7)CCATAAGAATTCCTGCAGGTTAACAGAGGACGGATTTCCTGAAGG

The following PCR conditions were used to amplify both pieces:

98° C. for 30 sec, 30× (98° C. for 10 sec, 55° C. for 20 sec, and 72° C.for 45 sec (thermolysin) or 72° C. for 80 sec (pHPLT)), followed by 72°C. for 5 min. The resulting PCR products were run on an E-gel(Invitrogen), excised, and purified with a gel extraction kit (Qiagen).In addition, a PCR overlap extension fusion (Ho, Gene, 15:51-59, 1989)was used to fuse the above gene fragments with High fidelity platinumTaq DNA polymerase (Invitrogen) using the following primers:

proT-EcoRI-FW: (SEQ ID NO: 7)CCATAAGAATTCCTGCAGGTTAACAGAGGACGGATTTCCTGAAGG proT-EcoRI-RV:(SEQ ID NO: 5) GTTAACCTGCAGGAATTCTTATTTCACCCCTACCGCATCAAAGGCC

The following conditions were used for these reactions:

94° C. for 2 min, 25× (94° C. for 30 sec, 55° C. for 30 sec, and 68° C.for 5 min) followed by 68° C. for 5 min. The resulting PCR fusionproduct was run on an E-gel (Invitrogen), excised, and purified with agel extraction kit (Qiagen). The purified fusion product was cut (PstI)and self-ligated (T4 DNA Ligase, Invitrogen). A map of thepHPLT-thermolysin expression vector is provided in FIG. 3, while the DNAsequence of the pHPLT-thermolysin expression vector (SEQ ID NO:8) isprovided in FIG. 4.

The ligation mixture was used to transform B. subtilis SC6.1 (phenotype:ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE:(xylR,pxylA-comK).Transformation of B. subtilis SC6.1 strain was performed as described inWO 02/14490, incorporated herein by reference. Selective growth of B.subtilis transformants containing the pHPLT-thermolysin vector was donein shake flasks containing 25 ml MBD medium (a MOPS based definedmedium), with 20 mg/L neomycin. Culturing resulted in the production ofsecreted mature thermolysin enzyme having proteolytic activity. Gelanalysis was performed using NuPage Novex 10% Bis-Tris gels (Invitrogen,Catalog No. NP0301BOX). To prepare samples for analysis, 2 volumes ofsupernatant were mixed with 1 volume 1M HCl, 1 volume 4×LDS samplebuffer (Invitrogen, Catalog No. NP0007), and 1% PMSF (20 mg/ml), andsubsequently heated for 10 minutes at 70° C. Then, 25 μL of each samplewas loaded onto the gel, adjacent to 10 μL of SeeBlue plus 2 pre-stainedprotein standards (Invitrogen, Catalog No. LC5925). The results clearlydemonstrated that the thermolysin cloning strategy described in thisexample is suitable for production of active recombinant thermolysin inB. subtilis.

Example 3 Generation of Thermolysin Site Evaluation Libraries (SELs)

In this Example, methods used in the construction of thermolysin SELsare described. As previously indicated, the terms “thermolysin,”“bacillolysin,” “proteinase-T” and “PrT” are used interchangeablythroughout to refer to the neutral metalloprotease enzyme of G.caldoproteolyticus. The pHPLT-thermolysin vector (FIG. 3) contains thethermolysin expression cassette, which served as a template DNA for thesite evaluation libraries. Every thermolysin site evaluation librarycontains a collection of B. subtilis clones, all expressing a specificthermolysin variant. Each library contains B. subtilis clones, maximallyincluding 20 different variants. For example, thermolysin SEL 27contains variants in which the DNA triplet coding for tyrosine atposition 27 of the mature thermolysin enzyme is replaced by another DNAtriplet encoding: Alanine, Aspartic acid, Cysteine, Glutamic acid,Phenylalanine, Glycine, Histidine, Isoleucine, Lysine, Leucine,Methionine, Asparagine, Proline, Glutamine, Arginine, Serine, Threonine,Valine, Tryptophan or Tyrosine.

Briefly, DNA triplets of specific positions in the DNA coding strand ofthe mature thermolysin are replaced. The mutated thermolysin fragmentsare subsequently ligated to pHPLT. The pHPLT-thermolysin variantplasmids are used to transform B. subtilis SC6.1 The production of prtvariants was done using the gene synthesis products and services ofSloning BioTechnology GmbH (Puchheim, Germany). The specific mutation ofeach variant was confirmed by DNA sequencing.

Example 4 Preparation of Crude Thermolysin Samples

The thermolysin (also referred to as Proteinase-T or PrT) variantproteins were produced by culturing the B. subtilis transformants in 96well MTP at 37° C. for 68 hours in MBD medium (a MOPS based definedmedium) including 10 mg/L neomycin. MBD medium was made essentially asknown in the art (See, Neidhardt et al., J Bacteriol, 119: 736-747,1974), except that NH₄Cl, FeSO₄, and CaCl₂ were omitted from the basemedium, 3 mM K₂HPO₄ was used, and the base medium was supplemented with60 mM urea, 75 g/L glucose, and 1% soytone. Also, the micronutrientswere made up as a 100× stock containing in one liter, 400 mg FeSO₄.7H₂O,100 mg MnSO₄.H₂O, 100 mg ZnSO4.7H₂O, 50 mg CuCl₂.2H₂O, 100 mgCoCl₂.6H₂O, 100 mg NaMoO₄.2H₂O, 100 mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂,and 10 ml of 0.5 M sodium citrate.

Example 5 Stability of Thermolysin in Heavy Duty Liquid (HDL) Detergent

Unilever detergent ALL Small and Mighty, P&G TIDE® Fresh Breeze, P&GTIDE® 2× Fresh Breeze were purchased from Walmart. The commerciallyavailable detergents were heated at 90° C. for 1 hour and then cooled toroom temperature, to inactivate the proteases in these cleaningcompositions. Thermolysin (also referred to as Proteinase-T or PrT) inlyophilized powder was purchased from Sigma, and dissolved in 100 mMTris pH 7 and 50% propylene glycol at 20 mg/ml. NprE was purified fromBacillus sp. supernatant through ion-exchange chromatography. To lml ofheat-treated detergent in an eppendorf tube, 800 □g of thermolysin orNprE was added. The tube was mixed well on a rocker for 15 min at roomtemperature, and then incubated at 25° C. or 32° C. At different timepoints, remaining proteinase activity was measured using an AGLA assayas described above in Example 1. Briefly, 10 □l of sample was diluted441 fold in AGLA buffer (50 mM MES, pH 6.5, 0.005% Tween 80, 2.5 mMCaCl₂), then 10 □l of diluted sample was added into 200 □l of AGLAsubstrate (2.4 mM Abz-AGLA-Nba in AGLA buffer). Excitation at 350 nm andemission at 415 nm was monitored for the first 100 seconds, and theinitial slope was recorded as enzyme activity. The enzyme activity wasplotted against time, and curves were fitted with exponential decay.

As shown in FIG. 5, thermolysin is 140 fold more stable than NprE inUnilever All Small & Mighty at room temperature. Similarly as shown inFIG. 6, thermolysin is 68 fold more stable than NprE in P&G TIDE® at 32°C., while FIG. 7 shows that thermolysin is 98 fold more stable than NprEin P&G TIDE® 2× at 32° C. Thus, thermolysin is much more stable thanNprE in Unilever detergent ALL (3×), P&G TIDE® 1× Fresh Breeze and P&GTIDE® 2× Fresh Breeze.

Example 6 Metalloproteinase Inhibitors can Improve Thermolysin Stabilityin Heavy Duty Liquid (HDL) Detergent

Zinc Chloride, Phosphoramidon, Galardin are known metalloproteinaseinhibitors. They were purchased from Sigma and dissolved in water orDMSO. Different concentrations of the inhibitors were premixed withthermolysin (also referred to as Proteinase-T or PrT) for 10 min at roomtemperature. Then the inhibitors were added into Unilever detergent ALLSmall and Mighty so that the final concentration of thermolysin was 800□g/ml in a total volume of 1 ml. At different time points, samples weretaken and precipitated with TCA. Briefly, 10 □l sample of detergent withenzyme was added into 500 □l of 0.2 N HCl on ice, and then 500 □l of 20%TCA was added. The tubes were mixed and incubated on ice for 20 min. Thepellet was collected and washed with 90% ice-cold acetone. The pelletwas dissolved in sample loading buffer (Invitrogen) for SDS-PAGEanalysis. As shown in FIG. 8, both 500 □M Phosphoramidon and 1 mMGalardin significantly stabilize thermolysin in detergent.

Example 7 Stain Removal Performance of Thermolysin Variants in a TIDE®2× Microswatch Assay

In this example, experiments were conducted to determine the stainremoval performance of various singly substituted thermolysin (alsoreferred to herein as Proteinase-T or PrT) variants. As described inExample 1, the stain cleaning performance of thermolysin variants wasdone utilizing a blood/milk/ink (BMI) microswatch assay. Briefly thecleaning performance of chosen single-substitution thermolysin variantswas assessed in a TIDE® 2× microswatch assay. Table 7-1 providesperformance indices for the tested variants (e.g., showing improvedperformance as compared to wild-type thermolysin enzyme). Those variantswith a performance index greater than 1 (PI>1) have improvedperformance. As indicated by these results, numerous variants havingsingle amino acid substitutions performed better than wild-type enzymein this assay system.

TABLE 7-1 Stain Removal For Variants With PI > 1 Variant PI T006G 1.13T006H 1.01 T006I 1.27 T006K 1.76 T006M 1.05 T006N 1.23 T006P 1.05 T006Q1.19 T006R 1.58 T006V 1.04 T006W 1.14 T006Y 1.06 V007F 1.08 V007H 1.32V007K 1.60 V007L 1.16 V007M 1.01 V007P 1.27 V007Q 1.20 V007R 1.53 V007T1.23 V007Y 1.11 T049G 1.01 T049H 1.25 T049I 1.24 T049K 1.01 T049L 1.25T049N 1.10 T049P 1.24 T049Q 1.30 T049W 1.10 A058I 1.04 A058P 1.10 A058R1.04 F063I 1.11 F063L 1.03 F063P 1.20 S065K 1.29 S065Y 1.05 Y075G 1.04Y075M 1.14 Y075T 1.01 Q128H 1.39 Q128I 1.34 Q128L 1.04 Q128M 1.10 Q128V1.07 Q128Y 1.13 Y151D 1.08 Y151E 1.11 Y151H 1.17 Y151K 1.03 Y151M 1.06Y151N 1.19 Y151Q 1.29 Y151R 1.75 Y151T 1.13 Y151V 1.25 Y151W 1.22 I156M1.11 I156R 1.22 I156T 1.03 I156W 1.16 G196R 1.13 Q273I 1.18 Q273P 1.13Q273Y 1.09 T278K 1.09 T278M 1.02 T278P 1.07 N280K 1.02 N280R 1.04

Example 8 Stability of Thermolysin Variants in TIDE® 2× Liquid Detergent

In this example, experiments were conducted to assess the stability ofvarious singly substituted thermolysin (also referred to herein asProteinase-T or PrT) variants in the presence of liquid detergent. Asdescribed in Example 1, the stability of thermolysin variants wasmeasured by determining the AGLA activity before and after incubation inthe presence of TIDE® 2× heavy duty liquid (HDL) detergent at anelevated temperature. The tables contain the relative stability valuescompared to wild-type thermolysin, which is the quotient of the variantresidual activity divided by the wild-type residual activity. A valuegreater than one indicates higher stability in the presence ofdetergent. In Table 8-1 and Table 8-2, data are provided showing therelative stability of single-substitution variants of thermolysinrelative to the stability of wild-type thermolysin in HDL detergent inthe presence and absence of DTPA.

TABLE 8-1 Stability Of Variants In 25% TIDE ® 2X With DTPA Variant PIT006A 1.01 T006C 1.03 T049D 1.05 T049I 1.01 T049L 1.02 T049M 1.02 T049N1.03 T049S 1.08 A056C 1.01 A056R 1.10 A056Y 1.05 A058S 1.02 S065C 1.05S065E 1.08 S065I 1.05 S065T 1.04 S065V 1.08 S065Y 1.05 Q128C 1.01 Q128I1.32 Q128M 1.06 Q128T 1.18 Q128V 1.45 Q128Y 1.09 Y151A 1.15 Y151C 1.25Y151D 1.12 Y151E 1.10 Y151H 1.11 Y151M 1.09 Y151N 1.25 Y151Q 1.03 Y151R1.26 Y151S 1.23 Y151T 1.18 Y151V 1.11 Y151W 1.02 I156E 1.58 I156H 1.21I156K 1.07 I156M 1.19 I156R 1.15 I156T 1.08 I156W 1.12 G196D 1.02 G196H1.19 Q273A 1.03 Q273N 1.25 Q273T 1.08 Q273W 1.05 Q273Y 1.05 T278C 1.05T278H 1.07 T278M 1.09 T278N 1.07 T278S 1.08 T278Y 1.05 N280E 1.13 N280I1.16 N280L 1.21 N280M 1.16 N280S 1.19

TABLE 8-2 Stability Of Variants In 25% TIDE ® 2X Without DTPA Variant PIT006C 1.07 T049D 1.28 T049N 1.07 T049Q 1.07 T049S 1.26 A056C 1.19 A056E1.07 A058C 1.01 A058E 1.24 Q061E 1.05 Q061M 1.01 S065C 1.14 S065D 1.20S065E 1.34 S065P 1.18 S065V 1.08 S065W 1.09 S065Y 1.05 Q128C 1.05 Q128I1.19 Q128M 1.09 Q128T 1.15 Q128V 1.20 Q128Y 1.05 Y151A 1.24 Y151C 1.09Y151N 1.05 Y151S 1.17 Y151T 1.10 I156E 1.09

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention, which are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

We claim:
 1. A composition comprising an isolated thermolysin and aneutral metalloprotease inhibitor, wherein said thermolysin is aGeobacillus thermolysin or a Bacillus thermolysin.
 2. The composition ofclaim 1, wherein said neutral metalloprotease inhibitor isphosphoramidon or galardin.
 3. The composition of claim 1, wherein saidGeobacillus is G. caldoproteolyticus or G. stearothermophilus.
 4. Thecomposition of claim 1, wherein said Bacillus is B. thermoproteolyticus.5. The composition of claim 1, wherein said thermolysin has at least 50%amino acid identity with the amino acid sequence set forth in SEQ IDNO:3.
 6. The composition of claim 1, wherein said thermolysin comprisesthe amino acid sequence set forth in SEQ ID NO:3.
 7. An isolatedthermolysin variant having improved stability and/or performance.
 8. Theisolated thermolysin variant of claim 7, wherein said thermolysinvariant is a Geobacillus thermolysin variant having an amino acidsequence comprising one or more substitutions at positions chosen frompositions equivalent to positions 6, 7, 49, 56, 58, 61, 63, 65, 75, 128,151, 156, 196, 273, 278, and 280 of the amino acid sequence set forth asSEQ ID NO:3.
 9. The isolated thermolysin variant of claim 8, whereinsaid one or more substitutions comprise one, two, three, four or fivesubstitutions at positions chosen from positions equivalent to positions6, 7, 49, 56, 58, 61, 63, 65, 75, 128, 151, 156, 196, 273, 278, and 280of the amino acid sequence set forth as SEQ ID NO:3.
 10. The isolatedthermolysin variant of claim 7, wherein said thermolysin variant is aGeobacillus thermolysin variant having an amino acid sequence comprisingone or more substitutions at positions chosen from positions equivalentto positions 4, 6, 7, 36, 49, 53, 56, 58, 61, 63, 65, 75, 85, 108, 128,129, 151, 156, 194, 195, 196, 261, 265, 273, 278, 280 and 297 of theamino acid sequence set forth as SEQ ID NO:3.
 11. The isolatedthermolysin variant of claim 10, wherein said one or more substitutionscomprise one, two, three, four or five substitutions at positions chosenfrom positions equivalent to positions 4, 6, 7, 36, 49, 53, 56, 58, 61,63, 65, 75, 85, 108, 128, 129, 151, 156, 194, 195, 196, 261, 265, 273,278, 280 and 297 of the amino acid sequence set forth as SEQ ID NO:3.12. The isolated thermolysin variant of claim 7, wherein saidthermolysin variant comprises one or more substitutions chosen fromsubstitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P, T006Q,T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P,V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N,T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K,S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y,Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V,Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K,T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M,T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V,Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H,Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S,T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q,T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E,S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y,Y151A, Y151C, Y151N, Y151S, Y151T, and I156E.
 13. The isolatedthermolysin variant of claim 12, wherein said one or more substitutionscomprise one, two, three, four or five substitutions chosen fromsubstitutions T006G, T006H, T006I, T006K, T006M, T006N, T006P, T006Q,T006R, T006V, T006W, T006Y, V007F, V007H, V007K, V007L, V007M, V007P,V007Q, V007R, V007T, V007Y, T049G, T049H, T049I, T049K, T049L, T049N,T049P, T049Q, T049W, A058I, A058P, A058R, F063I, F063L, F063P, S065K,S065Y, Y075G, Y075M, Y075T, Q128H, Q128I, Q128L, Q128M, Q128V, Q128Y,Y151D, Y151E, Y151H, Y151K, Y151M, Y151N, Y151Q, Y151R, Y151T, Y151V,Y151W, I156M, I156R, I156T, I156W, G196R, Q273I, Q273P, Q273Y, T278K,T278M, T278P, N280K, N280R, T006A, T006C, T049D, T049I, T049L, T049M,T049N, T049S, A056C, A056R, A056Y, A058S, S065C, S065E, S0651, S065T,S065V, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y, Y151A, Y151C,Y151D, Y151E, Y151H, Y151M, Y151N, Y151Q, Y151R, Y151S, Y151T, Y151V,Y151W, I156E, I156H, I156K, I156M, I156R, I156T, I156W, G196D, G196H,Q273A, Q273N, Q273T, Q273W, Q273Y, T278C, T278H, T278M, T278N, T278S,T278Y, N280E, N280I, N280L, N280M, N280S, T006C, T049D, T049N, T049Q,T049S, A056C, A056E, A058C, A058E, Q061E, Q061M, S065C, S065D, S065E,S065P, S065V, S065W, S065Y, Q128C, Q128I, Q128M, Q128T, Q128V, Q128Y,Y151A, Y151C, Y151N, Y151S, Y151T, and I156E.
 14. An isolatedthermolysin variant having an improvement in stability and/orperformance as compared to wild-type Geobacillus sp. thermolysin (e.g.,thermolysin comprising the amino acid sequence set forth as SEQ IDNO:3).
 15. The isolated thermolysin variant of claim 14, wherein saidimprovement comprises one or more of improved thermostability, improvedperformance under lower pH conditions, improved performance under higherpH conditions, and improved autolytic stability.
 16. A method forproducing an enzyme having thermolysin activity, comprising: i)transforming a host cell with an expression vector comprising apolynucleotide encoding a thermolysin variant having 50 to 99% aminoacid identity with the amino acid sequence set forth in SEQ ID NO:3; andii) cultivating said transformed host cell under conditions suitable forthe production of said thermolysin.
 17. The method of claim 16, whereinsaid method further comprises the step of harvesting the producedthermolysin.
 18. The method of claim 16, wherein said host cell is aBacillus species (e.g., B subtilis).
 19. A composition comprising atleast one thermolysin variant obtained from the recombinant Bacillus sp.host cell of claim
 18. 20. The composition of claim 7, wherein saidcomposition further comprises at least one calcium ion and/or zinc ion.21. The composition of claim 7, wherein said composition furthercomprises at least one stabilizer.
 22. The composition of claim 21,wherein said stabilizer is chosen from borax, glycerol, zinc ions,calcium ions, and calcium formate.
 23. The composition of claim 21,wherein said stabilizer is at least one competitive inhibitor thatstabilizes the at least one thermolysin in the presence of an anionicsurfactant.
 24. The composition of claim 7, wherein said composition isa cleaning composition.
 25. The composition of claim 24, wherein saidcleaning composition is a detergent.
 26. The composition of claim 7,further comprising at least one additional enzyme or enzyme derivativechosen from proteases, amylases, lipases, mannanases, pectinases,cutinases, oxidoreductases, hemicellulases, and cellulases.
 27. Thecomposition of claim 7, wherein said composition comprises at leastabout 0.0001 weight percent of said thermolysin variant.
 28. Thecomposition of claim 7, wherein said composition comprises from about0.001 to about 0.5 weight percent of said thermolysin variant.
 29. Thecomposition of claim 7, further comprising at least one adjunctingredient.
 30. The composition of claim 7, further comprising asufficient amount of a pH modifier to provide the composition with aneat pH of from about 3 to about 5, the composition being essentiallyfree of materials that hydrolyze at a pH of from about pH 3 to about pH5.
 31. The composition of claim 30, wherein said materials thathydrolyze at a pH of from about pH 3 to about pH 5 comprise at least onesurfactant.
 32. The composition of claim 31, wherein said surfactant isa sodium alkyl sulfate surfactant comprising an ethylene oxide moiety.33. The composition of claim 7, wherein said composition is a liquid.34. An animal feed composition comprising the isolated thermolysinvariant of claim
 7. 35. A textile processing composition comprising theisolated thermolysin variant of claim
 7. 36. A leather processingcomposition comprising the isolated thermolysin variant of claim
 7. 37.A method of cleaning, comprising the step of contacting a surface and/oran article comprising a fabric with a cleaning composition comprisingthe isolated thermolysin variant of claim
 7. 38. The method of claim 37,further comprising the step of rinsing the surface and/or material aftercontacting the surface or material with the cleaning composition.